US11725073B2 - Compositions and methods for liquid phase oligonucleotide synthesis - Google Patents
Compositions and methods for liquid phase oligonucleotide synthesis Download PDFInfo
- Publication number
- US11725073B2 US11725073B2 US17/562,714 US202117562714A US11725073B2 US 11725073 B2 US11725073 B2 US 11725073B2 US 202117562714 A US202117562714 A US 202117562714A US 11725073 B2 US11725073 B2 US 11725073B2
- Authority
- US
- United States
- Prior art keywords
- bioconjugate
- pvh
- alkyl
- optionally protected
- group
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
- 238000000034 method Methods 0.000 title claims abstract description 84
- 239000007791 liquid phase Substances 0.000 title claims abstract description 35
- 238000002515 oligonucleotide synthesis Methods 0.000 title claims abstract description 26
- 239000000203 mixture Substances 0.000 title description 7
- 239000002777 nucleoside Substances 0.000 claims abstract description 29
- 150000003833 nucleoside derivatives Chemical class 0.000 claims abstract description 26
- 108091034117 Oligonucleotide Proteins 0.000 claims abstract description 20
- 239000002904 solvent Substances 0.000 claims abstract description 20
- -1 (4-methoxyphenyl)diphenylmethyl Chemical group 0.000 claims description 64
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 claims description 57
- 125000004169 (C1-C6) alkyl group Chemical group 0.000 claims description 48
- 238000000502 dialysis Methods 0.000 claims description 33
- 125000002887 hydroxy group Chemical group [H]O* 0.000 claims description 25
- UYTPUPDQBNUYGX-UHFFFAOYSA-N guanine Chemical compound O=C1NC(N)=NC2=C1N=CN2 UYTPUPDQBNUYGX-UHFFFAOYSA-N 0.000 claims description 24
- 238000006243 chemical reaction Methods 0.000 claims description 23
- 125000006239 protecting group Chemical group 0.000 claims description 22
- 230000000903 blocking effect Effects 0.000 claims description 20
- 125000000623 heterocyclic group Chemical group 0.000 claims description 20
- 230000015572 biosynthetic process Effects 0.000 claims description 18
- 229920001577 copolymer Polymers 0.000 claims description 17
- 125000005843 halogen group Chemical group 0.000 claims description 15
- 239000012528 membrane Substances 0.000 claims description 15
- 125000002496 methyl group Chemical group [H]C([H])([H])* 0.000 claims description 15
- 239000004627 regenerated cellulose Substances 0.000 claims description 15
- WYURNTSHIVDZCO-UHFFFAOYSA-N Tetrahydrofuran Chemical compound C1CCOC1 WYURNTSHIVDZCO-UHFFFAOYSA-N 0.000 claims description 14
- ISAKRJDGNUQOIC-UHFFFAOYSA-N Uracil Chemical compound O=C1C=CNC(=O)N1 ISAKRJDGNUQOIC-UHFFFAOYSA-N 0.000 claims description 14
- OPTASPLRGRRNAP-UHFFFAOYSA-N cytosine Chemical compound NC=1C=CNC(=O)N=1 OPTASPLRGRRNAP-UHFFFAOYSA-N 0.000 claims description 14
- 125000003729 nucleotide group Chemical group 0.000 claims description 14
- RWQNBRDOKXIBIV-UHFFFAOYSA-N thymine Chemical compound CC1=CNC(=O)NC1=O RWQNBRDOKXIBIV-UHFFFAOYSA-N 0.000 claims description 14
- 229920000058 polyacrylate Polymers 0.000 claims description 13
- 229930024421 Adenine Natural products 0.000 claims description 12
- GFFGJBXGBJISGV-UHFFFAOYSA-N Adenine Chemical compound NC1=NC=NC2=C1N=CN2 GFFGJBXGBJISGV-UHFFFAOYSA-N 0.000 claims description 12
- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical compound ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 claims description 12
- ZMXDDKWLCZADIW-UHFFFAOYSA-N N,N-Dimethylformamide Chemical compound CN(C)C=O ZMXDDKWLCZADIW-UHFFFAOYSA-N 0.000 claims description 12
- 229960000643 adenine Drugs 0.000 claims description 12
- 229910052736 halogen Inorganic materials 0.000 claims description 11
- 229910052757 nitrogen Inorganic materials 0.000 claims description 11
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 claims description 11
- 238000001914 filtration Methods 0.000 claims description 10
- 239000001257 hydrogen Substances 0.000 claims description 10
- 229910052739 hydrogen Inorganic materials 0.000 claims description 10
- QJGQUHMNIGDVPM-UHFFFAOYSA-N nitrogen group Chemical group [N] QJGQUHMNIGDVPM-UHFFFAOYSA-N 0.000 claims description 10
- 125000001153 fluoro group Chemical group F* 0.000 claims description 9
- 125000000449 nitro group Chemical group [O-][N+](*)=O 0.000 claims description 9
- 125000004433 nitrogen atom Chemical group N* 0.000 claims description 9
- 229910052760 oxygen Inorganic materials 0.000 claims description 9
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 claims description 8
- 125000004093 cyano group Chemical group *C#N 0.000 claims description 8
- 125000004185 ester group Chemical group 0.000 claims description 8
- 125000001997 phenyl group Chemical group [H]C1=C([H])C([H])=C(*)C([H])=C1[H] 0.000 claims description 8
- JLCPHMBAVCMARE-UHFFFAOYSA-N [3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-[[3-[[3-[[3-[[3-[[3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-hydroxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methyl [5-(6-aminopurin-9-yl)-2-(hydroxymethyl)oxolan-3-yl] hydrogen phosphate Polymers Cc1cn(C2CC(OP(O)(=O)OCC3OC(CC3OP(O)(=O)OCC3OC(CC3O)n3cnc4c3nc(N)[nH]c4=O)n3cnc4c3nc(N)[nH]c4=O)C(COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3CO)n3cnc4c(N)ncnc34)n3ccc(N)nc3=O)n3cnc4c(N)ncnc34)n3ccc(N)nc3=O)n3ccc(N)nc3=O)n3ccc(N)nc3=O)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cc(C)c(=O)[nH]c3=O)n3cc(C)c(=O)[nH]c3=O)n3ccc(N)nc3=O)n3cc(C)c(=O)[nH]c3=O)n3cnc4c3nc(N)[nH]c4=O)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)O2)c(=O)[nH]c1=O JLCPHMBAVCMARE-UHFFFAOYSA-N 0.000 claims description 7
- 229940104302 cytosine Drugs 0.000 claims description 7
- 150000002367 halogens Chemical class 0.000 claims description 7
- 229920002401 polyacrylamide Polymers 0.000 claims description 7
- 229910052717 sulfur Inorganic materials 0.000 claims description 7
- YLQBMQCUIZJEEH-UHFFFAOYSA-N tetrahydrofuran Natural products C=1C=COC=1 YLQBMQCUIZJEEH-UHFFFAOYSA-N 0.000 claims description 7
- 229940113082 thymine Drugs 0.000 claims description 7
- 229940035893 uracil Drugs 0.000 claims description 7
- 150000002148 esters Chemical class 0.000 claims description 6
- 238000002955 isolation Methods 0.000 claims description 6
- OJMIONKXNSYLSR-UHFFFAOYSA-N phosphorous acid Chemical group OP(O)O OJMIONKXNSYLSR-UHFFFAOYSA-N 0.000 claims description 6
- UFHFLCQGNIYNRP-UHFFFAOYSA-N Hydrogen Chemical compound [H][H] UFHFLCQGNIYNRP-UHFFFAOYSA-N 0.000 claims description 5
- 125000002221 trityl group Chemical group [H]C1=C([H])C([H])=C([H])C([H])=C1C([*])(C1=C(C(=C(C(=C1[H])[H])[H])[H])[H])C1=C([H])C([H])=C([H])C([H])=C1[H] 0.000 claims description 5
- 239000003586 protic polar solvent Substances 0.000 claims description 4
- 239000011159 matrix material Substances 0.000 claims description 3
- 230000001590 oxidative effect Effects 0.000 claims description 3
- HXJUTPCZVOIRIF-UHFFFAOYSA-N sulfolane Chemical compound O=S1(=O)CCCC1 HXJUTPCZVOIRIF-UHFFFAOYSA-N 0.000 claims description 3
- 125000006575 electron-withdrawing group Chemical group 0.000 claims description 2
- 238000000108 ultra-filtration Methods 0.000 claims description 2
- 150000002431 hydrogen Chemical class 0.000 claims 2
- 125000000020 sulfo group Chemical group O=S(=O)([*])O[H] 0.000 claims 1
- 229920000642 polymer Polymers 0.000 abstract description 24
- 125000000524 functional group Chemical group 0.000 abstract description 3
- 239000002585 base Substances 0.000 description 19
- NIXOWILDQLNWCW-UHFFFAOYSA-M Acrylate Chemical compound [O-]C(=O)C=C NIXOWILDQLNWCW-UHFFFAOYSA-M 0.000 description 16
- 238000003786 synthesis reaction Methods 0.000 description 16
- 125000004191 (C1-C6) alkoxy group Chemical group 0.000 description 15
- XKRFYHLGVUSROY-UHFFFAOYSA-N Argon Chemical compound [Ar] XKRFYHLGVUSROY-UHFFFAOYSA-N 0.000 description 14
- 125000000217 alkyl group Chemical group 0.000 description 13
- 239000000243 solution Substances 0.000 description 13
- UBTJZUKVKGZHAD-UPRLRBBYSA-N 1-[(2r,4s,5r)-5-[[bis(4-methoxyphenyl)-phenylmethoxy]methyl]-4-hydroxyoxolan-2-yl]-5-methylpyrimidine-2,4-dione Chemical compound C1=CC(OC)=CC=C1C(C=1C=CC(OC)=CC=1)(C=1C=CC=CC=1)OC[C@@H]1[C@@H](O)C[C@H](N2C(NC(=O)C(C)=C2)=O)O1 UBTJZUKVKGZHAD-UPRLRBBYSA-N 0.000 description 12
- KDCGOANMDULRCW-UHFFFAOYSA-N Purine Natural products N1=CNC2=NC=NC2=C1 KDCGOANMDULRCW-UHFFFAOYSA-N 0.000 description 12
- VLKZOEOYAKHREP-UHFFFAOYSA-N n-Hexane Chemical compound CCCCCC VLKZOEOYAKHREP-UHFFFAOYSA-N 0.000 description 12
- 125000004432 carbon atom Chemical group C* 0.000 description 9
- 238000011068 loading method Methods 0.000 description 9
- 125000004737 (C1-C6) haloalkoxy group Chemical group 0.000 description 8
- 125000000171 (C1-C6) haloalkyl group Chemical group 0.000 description 8
- 125000002103 4,4'-dimethoxytriphenylmethyl group Chemical group [H]C1=C([H])C([H])=C(C([H])=C1[H])C(*)(C1=C([H])C([H])=C(OC([H])([H])[H])C([H])=C1[H])C1=C([H])C([H])=C(OC([H])([H])[H])C([H])=C1[H] 0.000 description 7
- CZPWVGJYEJSRLH-UHFFFAOYSA-N Pyrimidine Chemical compound C1=CN=CN=C1 CZPWVGJYEJSRLH-UHFFFAOYSA-N 0.000 description 7
- 229910052786 argon Inorganic materials 0.000 description 7
- 125000004122 cyclic group Chemical group 0.000 description 7
- 229910052751 metal Inorganic materials 0.000 description 7
- 239000002184 metal Substances 0.000 description 7
- 239000000178 monomer Substances 0.000 description 7
- 238000011084 recovery Methods 0.000 description 7
- SPEUIVXLLWOEMJ-UHFFFAOYSA-N 1,1-dimethoxyethane Chemical compound COC(C)OC SPEUIVXLLWOEMJ-UHFFFAOYSA-N 0.000 description 6
- HRPVXLWXLXDGHG-UHFFFAOYSA-N Acrylamide Chemical group NC(=O)C=C HRPVXLWXLXDGHG-UHFFFAOYSA-N 0.000 description 6
- 239000000385 dialysis solution Substances 0.000 description 6
- 125000005842 heteroatom Chemical group 0.000 description 6
- 150000007523 nucleic acids Chemical class 0.000 description 6
- 239000007787 solid Substances 0.000 description 6
- WFDIJRYMOXRFFG-UHFFFAOYSA-N Acetic anhydride Chemical compound CC(=O)OC(C)=O WFDIJRYMOXRFFG-UHFFFAOYSA-N 0.000 description 5
- 125000003118 aryl group Chemical group 0.000 description 5
- 238000005516 engineering process Methods 0.000 description 5
- 108020004707 nucleic acids Proteins 0.000 description 5
- 102000039446 nucleic acids Human genes 0.000 description 5
- 230000008569 process Effects 0.000 description 5
- 239000000047 product Substances 0.000 description 5
- 229920005604 random copolymer Polymers 0.000 description 5
- 239000007790 solid phase Substances 0.000 description 5
- ASJSAQIRZKANQN-CRCLSJGQSA-N 2-deoxy-D-ribose Chemical compound OC[C@@H](O)[C@@H](O)CC=O ASJSAQIRZKANQN-CRCLSJGQSA-N 0.000 description 4
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 description 4
- 125000004429 atom Chemical group 0.000 description 4
- 125000000484 butyl group Chemical group [H]C([*])([H])C([H])([H])C([H])([H])C([H])([H])[H] 0.000 description 4
- 229910052799 carbon Inorganic materials 0.000 description 4
- 150000001875 compounds Chemical class 0.000 description 4
- 125000001495 ethyl group Chemical group [H]C([H])([H])C([H])([H])* 0.000 description 4
- 125000004435 hydrogen atom Chemical group [H]* 0.000 description 4
- 150000002739 metals Chemical class 0.000 description 4
- 238000001728 nano-filtration Methods 0.000 description 4
- 108091033319 polynucleotide Proteins 0.000 description 4
- 102000040430 polynucleotide Human genes 0.000 description 4
- 239000002157 polynucleotide Substances 0.000 description 4
- 125000001436 propyl group Chemical group [H]C([*])([H])C([H])([H])C([H])([H])[H] 0.000 description 4
- 238000000425 proton nuclear magnetic resonance spectrum Methods 0.000 description 4
- 238000000746 purification Methods 0.000 description 4
- 125000000548 ribosyl group Chemical group C1([C@H](O)[C@H](O)[C@H](O1)CO)* 0.000 description 4
- 150000003384 small molecules Chemical class 0.000 description 4
- GXJPHSMOOJJMKN-UHFFFAOYSA-N (2,3,5,6-tetrafluorophenyl) prop-2-enoate Chemical compound FC1=CC(F)=C(F)C(OC(=O)C=C)=C1F GXJPHSMOOJJMKN-UHFFFAOYSA-N 0.000 description 3
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical group [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 3
- QGJOPFRUJISHPQ-UHFFFAOYSA-N Carbon disulfide Chemical compound S=C=S QGJOPFRUJISHPQ-UHFFFAOYSA-N 0.000 description 3
- 238000005481 NMR spectroscopy Methods 0.000 description 3
- WEVYAHXRMPXWCK-FIBGUPNXSA-N acetonitrile-d3 Chemical compound [2H]C([2H])([2H])C#N WEVYAHXRMPXWCK-FIBGUPNXSA-N 0.000 description 3
- 230000008901 benefit Effects 0.000 description 3
- 229920001400 block copolymer Polymers 0.000 description 3
- 150000001768 cations Chemical class 0.000 description 3
- 239000003153 chemical reaction reagent Substances 0.000 description 3
- 125000001309 chloro group Chemical group Cl* 0.000 description 3
- 125000004051 hexyl group Chemical group [H]C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])* 0.000 description 3
- 229920001519 homopolymer Polymers 0.000 description 3
- 238000003760 magnetic stirring Methods 0.000 description 3
- 229940088644 n,n-dimethylacrylamide Drugs 0.000 description 3
- YLGYACDQVQQZSW-UHFFFAOYSA-N n,n-dimethylprop-2-enamide Chemical compound CN(C)C(=O)C=C YLGYACDQVQQZSW-UHFFFAOYSA-N 0.000 description 3
- 239000002773 nucleotide Substances 0.000 description 3
- 125000001147 pentyl group Chemical group C(CCCC)* 0.000 description 3
- 125000002467 phosphate group Chemical group [H]OP(=O)(O[H])O[*] 0.000 description 3
- 241000894007 species Species 0.000 description 3
- 238000003756 stirring Methods 0.000 description 3
- LBUJPTNKIBCYBY-UHFFFAOYSA-N 1,2,3,4-tetrahydroquinoline Chemical compound C1=CC=C2CCCNC2=C1 LBUJPTNKIBCYBY-UHFFFAOYSA-N 0.000 description 2
- WQADWIOXOXRPLN-UHFFFAOYSA-N 1,3-dithiane Chemical compound C1CSCSC1 WQADWIOXOXRPLN-UHFFFAOYSA-N 0.000 description 2
- 238000005160 1H NMR spectroscopy Methods 0.000 description 2
- FZWGECJQACGGTI-UHFFFAOYSA-N 2-amino-7-methyl-1,7-dihydro-6H-purin-6-one Chemical compound NC1=NC(O)=C2N(C)C=NC2=N1 FZWGECJQACGGTI-UHFFFAOYSA-N 0.000 description 2
- 125000001731 2-cyanoethyl group Chemical group [H]C([H])(*)C([H])([H])C#N 0.000 description 2
- OIVLITBTBDPEFK-UHFFFAOYSA-N 5,6-dihydrouracil Chemical compound O=C1CCNC(=O)N1 OIVLITBTBDPEFK-UHFFFAOYSA-N 0.000 description 2
- PEHVGBZKEYRQSX-UHFFFAOYSA-N 7-deaza-adenine Chemical compound NC1=NC=NC2=C1C=CN2 PEHVGBZKEYRQSX-UHFFFAOYSA-N 0.000 description 2
- LRFVTYWOQMYALW-UHFFFAOYSA-N 9H-xanthine Chemical compound O=C1NC(=O)NC2=C1NC=N2 LRFVTYWOQMYALW-UHFFFAOYSA-N 0.000 description 2
- 125000001313 C5-C10 heteroaryl group Chemical group 0.000 description 2
- 108020004414 DNA Proteins 0.000 description 2
- XOBKSJJDNFUZPF-UHFFFAOYSA-N Methoxyethane Chemical compound CCOC XOBKSJJDNFUZPF-UHFFFAOYSA-N 0.000 description 2
- PYMYPHUHKUWMLA-LMVFSUKVSA-N Ribose Natural products OC[C@@H](O)[C@@H](O)[C@@H](O)C=O PYMYPHUHKUWMLA-LMVFSUKVSA-N 0.000 description 2
- 239000004809 Teflon Substances 0.000 description 2
- 229920006362 Teflon® Polymers 0.000 description 2
- DHKHKXVYLBGOIT-UHFFFAOYSA-N acetaldehyde Diethyl Acetal Natural products CCOC(C)OCC DHKHKXVYLBGOIT-UHFFFAOYSA-N 0.000 description 2
- NIXOWILDQLNWCW-UHFFFAOYSA-N acrylic acid group Chemical group C(C=C)(=O)O NIXOWILDQLNWCW-UHFFFAOYSA-N 0.000 description 2
- 125000002015 acyclic group Chemical group 0.000 description 2
- 125000003545 alkoxy group Chemical group 0.000 description 2
- HMFHBZSHGGEWLO-UHFFFAOYSA-N alpha-D-Furanose-Ribose Natural products OCC1OC(O)C(O)C1O HMFHBZSHGGEWLO-UHFFFAOYSA-N 0.000 description 2
- 238000004458 analytical method Methods 0.000 description 2
- 125000001797 benzyl group Chemical group [H]C1=C([H])C([H])=C(C([H])=C1[H])C([H])([H])* 0.000 description 2
- RYYVLZVUVIJVGH-UHFFFAOYSA-N caffeine Chemical compound CN1C(=O)N(C)C(=O)C2=C1N=CN2C RYYVLZVUVIJVGH-UHFFFAOYSA-N 0.000 description 2
- XXBDWLFCJWSEKW-UHFFFAOYSA-N dimethylbenzylamine Chemical compound CN(C)CC1=CC=CC=C1 XXBDWLFCJWSEKW-UHFFFAOYSA-N 0.000 description 2
- 230000007062 hydrolysis Effects 0.000 description 2
- 238000006460 hydrolysis reaction Methods 0.000 description 2
- FDGQSTZJBFJUBT-UHFFFAOYSA-N hypoxanthine Chemical compound O=C1NC=NC2=C1NC=N2 FDGQSTZJBFJUBT-UHFFFAOYSA-N 0.000 description 2
- 125000000959 isobutyl group Chemical group [H]C([H])([H])C([H])(C([H])([H])[H])C([H])([H])* 0.000 description 2
- DRAVOWXCEBXPTN-UHFFFAOYSA-N isoguanine Chemical compound NC1=NC(=O)NC2=C1NC=N2 DRAVOWXCEBXPTN-UHFFFAOYSA-N 0.000 description 2
- 125000001449 isopropyl group Chemical group [H]C([H])([H])C([H])(*)C([H])([H])[H] 0.000 description 2
- 239000007788 liquid Substances 0.000 description 2
- 238000004519 manufacturing process Methods 0.000 description 2
- 239000000463 material Substances 0.000 description 2
- NSPJNIDYTSSIIY-UHFFFAOYSA-N methoxy(methoxymethoxy)methane Chemical compound COCOCOC NSPJNIDYTSSIIY-UHFFFAOYSA-N 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 239000001301 oxygen Substances 0.000 description 2
- 125000004430 oxygen atom Chemical group O* 0.000 description 2
- 150000002972 pentoses Chemical class 0.000 description 2
- 238000010647 peptide synthesis reaction Methods 0.000 description 2
- 108090000765 processed proteins & peptides Proteins 0.000 description 2
- 230000000717 retained effect Effects 0.000 description 2
- 239000000126 substance Substances 0.000 description 2
- 125000001424 substituent group Chemical group 0.000 description 2
- 239000000725 suspension Substances 0.000 description 2
- 238000001308 synthesis method Methods 0.000 description 2
- ILMRJRBKQSSXGY-UHFFFAOYSA-N tert-butyl(dimethyl)silicon Chemical group C[Si](C)C(C)(C)C ILMRJRBKQSSXGY-UHFFFAOYSA-N 0.000 description 2
- YAPQBXQYLJRXSA-UHFFFAOYSA-N theobromine Chemical compound CN1C(=O)NC(=O)C2=C1N=CN2C YAPQBXQYLJRXSA-UHFFFAOYSA-N 0.000 description 2
- 125000004178 (C1-C4) alkyl group Chemical group 0.000 description 1
- 125000006727 (C1-C6) alkenyl group Chemical group 0.000 description 1
- 125000006700 (C1-C6) alkylthio group Chemical group 0.000 description 1
- 125000006728 (C1-C6) alkynyl group Chemical group 0.000 description 1
- 125000006716 (C1-C6) heteroalkyl group Chemical group 0.000 description 1
- 125000006714 (C3-C10) heterocyclyl group Chemical group 0.000 description 1
- VDFVNEFVBPFDSB-UHFFFAOYSA-N 1,3-dioxane Chemical compound C1COCOC1 VDFVNEFVBPFDSB-UHFFFAOYSA-N 0.000 description 1
- IGERFAHWSHDDHX-UHFFFAOYSA-N 1,3-dioxanyl Chemical group [CH]1OCCCO1 IGERFAHWSHDDHX-UHFFFAOYSA-N 0.000 description 1
- 125000006091 1,3-dioxolane group Chemical class 0.000 description 1
- JPRPJUMQRZTTED-UHFFFAOYSA-N 1,3-dioxolanyl Chemical group [CH]1OCCO1 JPRPJUMQRZTTED-UHFFFAOYSA-N 0.000 description 1
- IMLSAISZLJGWPP-UHFFFAOYSA-N 1,3-dithiolane Chemical compound C1CSCS1 IMLSAISZLJGWPP-UHFFFAOYSA-N 0.000 description 1
- FLOJNXXFMHCMMR-UHFFFAOYSA-N 1,3-dithiolanyl Chemical group [CH]1SCCS1 FLOJNXXFMHCMMR-UHFFFAOYSA-N 0.000 description 1
- KFHQOZXAFUKFNB-UHFFFAOYSA-N 1,3-oxathiolanyl Chemical group [CH]1OCCS1 KFHQOZXAFUKFNB-UHFFFAOYSA-N 0.000 description 1
- 125000005940 1,4-dioxanyl group Chemical group 0.000 description 1
- HUHXLHLWASNVDB-UHFFFAOYSA-N 2-(oxan-2-yloxy)oxane Chemical class O1CCCCC1OC1OCCCC1 HUHXLHLWASNVDB-UHFFFAOYSA-N 0.000 description 1
- WYGWHHGCAGTUCH-UHFFFAOYSA-N 2-[(2-cyano-4-methylpentan-2-yl)diazenyl]-2,4-dimethylpentanenitrile Chemical compound CC(C)CC(C)(C#N)N=NC(C)(C#N)CC(C)C WYGWHHGCAGTUCH-UHFFFAOYSA-N 0.000 description 1
- JDQDSEVNMTYMOC-UHFFFAOYSA-N 3-methylbenzenesulfonic acid Chemical compound CC1=CC=CC(S(O)(=O)=O)=C1 JDQDSEVNMTYMOC-UHFFFAOYSA-N 0.000 description 1
- 125000005986 4-piperidonyl group Chemical group 0.000 description 1
- LRSASMSXMSNRBT-UHFFFAOYSA-N 5-methylcytosine Chemical compound CC1=CNC(=O)N=C1N LRSASMSXMSNRBT-UHFFFAOYSA-N 0.000 description 1
- LOSIULRWFAEMFL-UHFFFAOYSA-N 7-deazaguanine Chemical compound O=C1NC(N)=NC2=C1CC=N2 LOSIULRWFAEMFL-UHFFFAOYSA-N 0.000 description 1
- DCERHCFNWRGHLK-UHFFFAOYSA-N C[Si](C)C Chemical compound C[Si](C)C DCERHCFNWRGHLK-UHFFFAOYSA-N 0.000 description 1
- HMFHBZSHGGEWLO-SOOFDHNKSA-N D-ribofuranose Chemical compound OC[C@H]1OC(O)[C@H](O)[C@@H]1O HMFHBZSHGGEWLO-SOOFDHNKSA-N 0.000 description 1
- LCGLNKUTAGEVQW-UHFFFAOYSA-N Dimethyl ether Chemical class COC LCGLNKUTAGEVQW-UHFFFAOYSA-N 0.000 description 1
- NYHBQMYGNKIUIF-UUOKFMHZSA-N Guanosine Chemical compound C1=NC=2C(=O)NC(N)=NC=2N1[C@@H]1O[C@H](CO)[C@@H](O)[C@H]1O NYHBQMYGNKIUIF-UUOKFMHZSA-N 0.000 description 1
- UGQMRVRMYYASKQ-UHFFFAOYSA-N Hypoxanthine nucleoside Natural products OC1C(O)C(CO)OC1N1C(NC=NC2=O)=C2N=C1 UGQMRVRMYYASKQ-UHFFFAOYSA-N 0.000 description 1
- LPHGQDQBBGAPDZ-UHFFFAOYSA-N Isocaffeine Natural products CN1C(=O)N(C)C(=O)C2=C1N(C)C=N2 LPHGQDQBBGAPDZ-UHFFFAOYSA-N 0.000 description 1
- AFVFQIVMOAPDHO-UHFFFAOYSA-N Methanesulfonic acid Chemical compound CS(O)(=O)=O AFVFQIVMOAPDHO-UHFFFAOYSA-N 0.000 description 1
- 108091028043 Nucleic acid sequence Proteins 0.000 description 1
- VHFVDEXSJFKLDW-UHFFFAOYSA-N OS(S(O)(=O)=O)(S(O)(=O)=O)=O Chemical compound OS(S(O)(=O)=O)(S(O)(=O)=O)=O VHFVDEXSJFKLDW-UHFFFAOYSA-N 0.000 description 1
- 229910019142 PO4 Inorganic materials 0.000 description 1
- 239000004642 Polyimide Substances 0.000 description 1
- 239000004793 Polystyrene Substances 0.000 description 1
- 108020004511 Recombinant DNA Proteins 0.000 description 1
- 125000005631 S-sulfonamido group Chemical group 0.000 description 1
- NINIDFKCEFEMDL-UHFFFAOYSA-N Sulfur Chemical group [S] NINIDFKCEFEMDL-UHFFFAOYSA-N 0.000 description 1
- LEHOTFFKMJEONL-UHFFFAOYSA-N Uric Acid Chemical compound N1C(=O)NC(=O)C2=C1NC(=O)N2 LEHOTFFKMJEONL-UHFFFAOYSA-N 0.000 description 1
- TVWHNULVHGKJHS-UHFFFAOYSA-N Uric acid Natural products N1C(=O)NC(=O)C2NC(=O)NC21 TVWHNULVHGKJHS-UHFFFAOYSA-N 0.000 description 1
- 238000002835 absorbance Methods 0.000 description 1
- 125000002777 acetyl group Chemical group [H]C([H])([H])C(*)=O 0.000 description 1
- 238000005903 acid hydrolysis reaction Methods 0.000 description 1
- 125000000641 acridinyl group Chemical group C1(=CC=CC2=NC3=CC=CC=C3C=C12)* 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- 125000002252 acyl group Chemical group 0.000 description 1
- 229910052783 alkali metal Inorganic materials 0.000 description 1
- 150000001340 alkali metals Chemical class 0.000 description 1
- 229910052784 alkaline earth metal Inorganic materials 0.000 description 1
- 150000001342 alkaline earth metals Chemical class 0.000 description 1
- 125000004448 alkyl carbonyl group Chemical group 0.000 description 1
- 125000005600 alkyl phosphonate group Chemical group 0.000 description 1
- 125000006620 amino-(C1-C6) alkyl group Chemical group 0.000 description 1
- 125000005099 aryl alkyl carbonyl group Chemical group 0.000 description 1
- 125000005110 aryl thio group Chemical group 0.000 description 1
- 125000004104 aryloxy group Chemical group 0.000 description 1
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 1
- 125000002785 azepinyl group Chemical group 0.000 description 1
- 125000003236 benzoyl group Chemical group [H]C1=C([H])C([H])=C(C([H])=C1[H])C(*)=O 0.000 description 1
- 125000001584 benzyloxycarbonyl group Chemical group C(=O)(OCC1=CC=CC=C1)* 0.000 description 1
- 230000008033 biological extinction Effects 0.000 description 1
- 238000010504 bond cleavage reaction Methods 0.000 description 1
- 229960001948 caffeine Drugs 0.000 description 1
- VJEONQKOZGKCAK-UHFFFAOYSA-N caffeine Natural products CN1C(=O)N(C)C(=O)C2=C1C=CN2C VJEONQKOZGKCAK-UHFFFAOYSA-N 0.000 description 1
- 125000000609 carbazolyl group Chemical group C1(=CC=CC=2C3=CC=CC=C3NC12)* 0.000 description 1
- 125000004452 carbocyclyl group Chemical group 0.000 description 1
- 150000001721 carbon Chemical group 0.000 description 1
- 150000004649 carbonic acid derivatives Chemical class 0.000 description 1
- 125000002915 carbonyl group Chemical group [*:2]C([*:1])=O 0.000 description 1
- 229920002301 cellulose acetate Polymers 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 125000000259 cinnolinyl group Chemical group N1=NC(=CC2=CC=CC=C12)* 0.000 description 1
- 230000021615 conjugation Effects 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 239000005549 deoxyribonucleoside Substances 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- MHDVGSVTJDSBDK-UHFFFAOYSA-N dibenzyl ether Chemical class C=1C=CC=CC=1COCC1=CC=CC=C1 MHDVGSVTJDSBDK-UHFFFAOYSA-N 0.000 description 1
- 125000000723 dihydrobenzofuranyl group Chemical group O1C(CC2=C1C=CC=C2)* 0.000 description 1
- NAGJZTKCGNOGPW-UHFFFAOYSA-K dioxido-sulfanylidene-sulfido-$l^{5}-phosphane Chemical compound [O-]P([O-])([S-])=S NAGJZTKCGNOGPW-UHFFFAOYSA-K 0.000 description 1
- 125000005879 dioxolanyl group Chemical group 0.000 description 1
- 239000001177 diphosphate Substances 0.000 description 1
- XPPKVPWEQAFLFU-UHFFFAOYSA-J diphosphate(4-) Chemical compound [O-]P([O-])(=O)OP([O-])([O-])=O XPPKVPWEQAFLFU-UHFFFAOYSA-J 0.000 description 1
- 235000011180 diphosphates Nutrition 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- KPUWHANPEXNPJT-UHFFFAOYSA-N disiloxane Chemical class [SiH3]O[SiH3] KPUWHANPEXNPJT-UHFFFAOYSA-N 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 230000002349 favourable effect Effects 0.000 description 1
- 239000000835 fiber Substances 0.000 description 1
- 239000011521 glass Substances 0.000 description 1
- 239000003365 glass fiber Substances 0.000 description 1
- 238000004128 high performance liquid chromatography Methods 0.000 description 1
- 150000002430 hydrocarbons Chemical group 0.000 description 1
- 125000002632 imidazolidinyl group Chemical group 0.000 description 1
- 125000002636 imidazolinyl group Chemical group 0.000 description 1
- 238000010348 incorporation Methods 0.000 description 1
- 125000003387 indolinyl group Chemical group N1(CCC2=CC=CC=C12)* 0.000 description 1
- 239000003999 initiator Substances 0.000 description 1
- 230000010354 integration Effects 0.000 description 1
- 125000004594 isoindolinyl group Chemical group C1(NCC2=CC=CC=C12)* 0.000 description 1
- 125000003253 isopropoxy group Chemical group [H]C([H])([H])C([H])(O*)C([H])([H])[H] 0.000 description 1
- 125000003965 isoxazolidinyl group Chemical group 0.000 description 1
- 125000003971 isoxazolinyl group Chemical group 0.000 description 1
- 150000002605 large molecules Chemical class 0.000 description 1
- 229920002521 macromolecule Polymers 0.000 description 1
- 238000004949 mass spectrometry Methods 0.000 description 1
- 229910000000 metal hydroxide Inorganic materials 0.000 description 1
- 150000004692 metal hydroxides Chemical class 0.000 description 1
- FQPSGWSUVKBHSU-UHFFFAOYSA-N methacrylamide Chemical group CC(=C)C(N)=O FQPSGWSUVKBHSU-UHFFFAOYSA-N 0.000 description 1
- 125000005397 methacrylic acid ester group Chemical group 0.000 description 1
- 125000000956 methoxy group Chemical group [H]C([H])([H])O* 0.000 description 1
- TUDYPXFSYJRWDP-UHFFFAOYSA-N methoxy methyl carbonate Chemical compound COOC(=O)OC TUDYPXFSYJRWDP-UHFFFAOYSA-N 0.000 description 1
- UPKVNYSOEWZBCU-UHFFFAOYSA-N mismatched oligo Chemical group O=C1NC(=O)C(C)=CN1C1OC(COP(O)(=S)OC2C(OC(C2)N2C3=C(C(NC(N)=N3)=O)N=C2)COP(O)(=S)OC2C(OC(C2)N2C(N=C(N)C=C2)=O)COP(O)(=S)OC2C(OC(C2)N2C3=NC=NC(N)=C3N=C2)COP(O)(=S)OC2C(OC(C2)N2C(NC(=O)C(C)=C2)=O)COP(O)(=S)OC2C(OC(C2)N2C3=C(C(NC(N)=N3)=O)N=C2)COP(O)(=S)OC2C(OC(C2)N2C(N=C(N)C=C2)=O)COP(O)(=S)OC2C(OC(C2)N2C3=NC=NC(N)=C3N=C2)COP(O)(=S)OC2C(OC(C2)N2C(NC(=O)C(C)=C2)=O)COP(O)(=S)OC2C(OC(C2)N2C3=C(C(NC(N)=N3)=O)N=C2)COP(O)(=S)OC2C(OC(C2)N2C(N=C(N)C=C2)=O)COP(O)(=S)OC2C(OC(C2)N2C3=NC=NC(N)=C3N=C2)COP(O)(=S)OC2C(OC(C2)N2C(NC(=O)C(C)=C2)=O)COP(O)(=S)OC2C(OC(C2)N2C3=C(C(NC(N)=N3)=O)N=C2)COP(O)(=S)OC2C(OC(C2)N2C(N=C(N)C=C2)=O)COP(O)(=S)OC2C(OC(C2)N2C3=NC=NC(N)=C3N=C2)COP(O)(=S)OC2C(OC(C2)N2C(NC(=O)C(C)=C2)=O)COP(O)(=S)OC2C(OC(C2)N2C3=C(C(NC(N)=N3)=O)N=C2)COP(O)(=S)OC2C(OC(C2)N2C(N=C(N)C=C2)=O)COP(O)(=S)OC2C(OC(C2)N2C3=NC=NC(N)=C3N=C2)COP(O)(=S)OC2C(OC(C2)N2C(NC(=O)C(C)=C2)=O)COP(O)(=S)OC2C(OC(C2)N2C3=C(C(NC(N)=N3)=O)N=C2)COP(O)(=S)OC2C(OC(C2)N2C(N=C(N)C=C2)=O)COP(O)(=S)OC2C(OC(C2)N2C3=NC=NC(N)=C3N=C2)CO)C(OP(O)(=S)OCC2C(CC(O2)N2C3=NC=NC(N)=C3N=C2)O)C1 UPKVNYSOEWZBCU-UHFFFAOYSA-N 0.000 description 1
- 150000004712 monophosphates Chemical class 0.000 description 1
- 125000002757 morpholinyl group Chemical group 0.000 description 1
- 125000004108 n-butyl group Chemical group [H]C([H])([H])C([H])([H])C([H])([H])C([H])([H])* 0.000 description 1
- 125000006574 non-aromatic ring group Chemical group 0.000 description 1
- 230000000269 nucleophilic effect Effects 0.000 description 1
- 229940127073 nucleoside analogue Drugs 0.000 description 1
- 150000002894 organic compounds Chemical class 0.000 description 1
- 239000003960 organic solvent Substances 0.000 description 1
- 125000000160 oxazolidinyl group Chemical group 0.000 description 1
- 125000005968 oxazolinyl group Chemical group 0.000 description 1
- 125000003551 oxepanyl group Chemical group 0.000 description 1
- 125000000466 oxiranyl group Chemical group 0.000 description 1
- 125000004043 oxo group Chemical group O=* 0.000 description 1
- HXNFUBHNUDHIGC-UHFFFAOYSA-N oxypurinol Chemical compound O=C1NC(=O)N=C2NNC=C21 HXNFUBHNUDHIGC-UHFFFAOYSA-N 0.000 description 1
- 239000002245 particle Substances 0.000 description 1
- UYWQUFXKFGHYNT-UHFFFAOYSA-N phenylmethyl ester of formic acid Natural products O=COCC1=CC=CC=C1 UYWQUFXKFGHYNT-UHFFFAOYSA-N 0.000 description 1
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 description 1
- 239000010452 phosphate Substances 0.000 description 1
- 150000004713 phosphodiesters Chemical class 0.000 description 1
- PTMHPRAIXMAOOB-UHFFFAOYSA-L phosphoramidate Chemical compound NP([O-])([O-])=O PTMHPRAIXMAOOB-UHFFFAOYSA-L 0.000 description 1
- 125000004193 piperazinyl group Chemical group 0.000 description 1
- 125000003386 piperidinyl group Chemical group 0.000 description 1
- 229920001721 polyimide Polymers 0.000 description 1
- 238000006116 polymerization reaction Methods 0.000 description 1
- 229920002223 polystyrene Polymers 0.000 description 1
- 239000005373 porous glass Substances 0.000 description 1
- 239000002244 precipitate Substances 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- IGFXRKMLLMBKSA-UHFFFAOYSA-N purine Chemical compound N1=C[N]C2=NC=NC2=C1 IGFXRKMLLMBKSA-UHFFFAOYSA-N 0.000 description 1
- 125000003072 pyrazolidinyl group Chemical group 0.000 description 1
- 125000002755 pyrazolinyl group Chemical group 0.000 description 1
- 125000000719 pyrrolidinyl group Chemical group 0.000 description 1
- 125000004929 pyrrolidonyl group Chemical group N1(C(CCC1)=O)* 0.000 description 1
- 239000011541 reaction mixture Substances 0.000 description 1
- 238000012552 review Methods 0.000 description 1
- 239000002342 ribonucleoside Substances 0.000 description 1
- 229920002477 rna polymer Polymers 0.000 description 1
- 229920006395 saturated elastomer Polymers 0.000 description 1
- 230000007017 scission Effects 0.000 description 1
- 125000002914 sec-butyl group Chemical group [H]C([H])([H])C([H])([H])C([H])(*)C([H])([H])[H] 0.000 description 1
- 229910052708 sodium Inorganic materials 0.000 description 1
- 239000011734 sodium Substances 0.000 description 1
- 238000001179 sorption measurement Methods 0.000 description 1
- 238000002798 spectrophotometry method Methods 0.000 description 1
- 238000001228 spectrum Methods 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- KDYFGRWQOYBRFD-UHFFFAOYSA-L succinate(2-) Chemical compound [O-]C(=O)CCC([O-])=O KDYFGRWQOYBRFD-UHFFFAOYSA-L 0.000 description 1
- PXQLVRUNWNTZOS-UHFFFAOYSA-N sulfanyl Chemical class [SH] PXQLVRUNWNTZOS-UHFFFAOYSA-N 0.000 description 1
- 150000003871 sulfonates Chemical class 0.000 description 1
- 125000000472 sulfonyl group Chemical group *S(*)(=O)=O 0.000 description 1
- 239000011593 sulfur Substances 0.000 description 1
- 239000006228 supernatant Substances 0.000 description 1
- 125000004213 tert-butoxy group Chemical group [H]C([H])([H])C(O*)(C([H])([H])[H])C([H])([H])[H] 0.000 description 1
- 125000000999 tert-butyl group Chemical group [H]C([H])([H])C(*)(C([H])([H])[H])C([H])([H])[H] 0.000 description 1
- 125000003718 tetrahydrofuranyl group Chemical group 0.000 description 1
- 125000001412 tetrahydropyranyl group Chemical group 0.000 description 1
- 125000003507 tetrahydrothiofenyl group Chemical group 0.000 description 1
- 125000004632 tetrahydrothiopyranyl group Chemical group S1C(CCCC1)* 0.000 description 1
- 229960004559 theobromine Drugs 0.000 description 1
- 125000001984 thiazolidinyl group Chemical group 0.000 description 1
- 125000002769 thiazolinyl group Chemical group 0.000 description 1
- 125000001583 thiepanyl group Chemical group 0.000 description 1
- 125000003396 thiol group Chemical group [H]S* 0.000 description 1
- 125000004568 thiomorpholinyl group Chemical group 0.000 description 1
- RYYWUUFWQRZTIU-UHFFFAOYSA-K thiophosphate Chemical compound [O-]P([O-])([O-])=S RYYWUUFWQRZTIU-UHFFFAOYSA-K 0.000 description 1
- JOXIMZWYDAKGHI-UHFFFAOYSA-M toluene-4-sulfonate Chemical compound CC1=CC=C(S([O-])(=O)=O)C=C1 JOXIMZWYDAKGHI-UHFFFAOYSA-M 0.000 description 1
- 125000005039 triarylmethyl group Chemical group 0.000 description 1
- 125000000876 trifluoromethoxy group Chemical group FC(F)(F)O* 0.000 description 1
- 125000000025 triisopropylsilyl group Chemical group C(C)(C)[Si](C(C)C)(C(C)C)* 0.000 description 1
- 125000000026 trimethylsilyl group Chemical group [H]C([H])([H])[Si]([*])(C([H])([H])[H])C([H])([H])[H] 0.000 description 1
- 239000001226 triphosphate Substances 0.000 description 1
- 235000011178 triphosphate Nutrition 0.000 description 1
- UNXRWKVEANCORM-UHFFFAOYSA-N triphosphoric acid Chemical compound OP(O)(=O)OP(O)(=O)OP(O)(O)=O UNXRWKVEANCORM-UHFFFAOYSA-N 0.000 description 1
- 238000002371 ultraviolet--visible spectrum Methods 0.000 description 1
- 229940116269 uric acid Drugs 0.000 description 1
- 238000005292 vacuum distillation Methods 0.000 description 1
- 238000001291 vacuum drying Methods 0.000 description 1
- 125000000391 vinyl group Chemical group [H]C([*])=C([H])[H] 0.000 description 1
- 229920002554 vinyl polymer Polymers 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
- 239000002699 waste material Substances 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
- 229940075420 xanthine Drugs 0.000 description 1
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08F—MACROMOLECULAR COMPOUNDS OBTAINED BY REACTIONS ONLY INVOLVING CARBON-TO-CARBON UNSATURATED BONDS
- C08F220/00—Copolymers of compounds having one or more unsaturated aliphatic radicals, each having only one carbon-to-carbon double bond, and only one being terminated by only one carboxyl radical or a salt, anhydride ester, amide, imide or nitrile thereof
- C08F220/02—Monocarboxylic acids having less than ten carbon atoms; Derivatives thereof
- C08F220/10—Esters
- C08F220/22—Esters containing halogen
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
- A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
- A61K47/54—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound
- A61K47/549—Sugars, nucleosides, nucleotides or nucleic acids
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
- A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
- A61K47/56—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule
- A61K47/58—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained by reactions only involving carbon-to-carbon unsaturated bonds, e.g. poly[meth]acrylate, polyacrylamide, polystyrene, polyvinylpyrrolidone, polyvinylalcohol or polystyrene sulfonic acid resin
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
- C07H19/00—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
- C07H21/00—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
- C07H21/02—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids with ribosyl as saccharide radical
-
- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08F—MACROMOLECULAR COMPOUNDS OBTAINED BY REACTIONS ONLY INVOLVING CARBON-TO-CARBON UNSATURATED BONDS
- C08F220/00—Copolymers of compounds having one or more unsaturated aliphatic radicals, each having only one carbon-to-carbon double bond, and only one being terminated by only one carboxyl radical or a salt, anhydride ester, amide, imide or nitrile thereof
- C08F220/02—Monocarboxylic acids having less than ten carbon atoms; Derivatives thereof
- C08F220/10—Esters
- C08F220/22—Esters containing halogen
- C08F220/24—Esters containing halogen containing perhaloalkyl radicals
-
- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08F—MACROMOLECULAR COMPOUNDS OBTAINED BY REACTIONS ONLY INVOLVING CARBON-TO-CARBON UNSATURATED BONDS
- C08F8/00—Chemical modification by after-treatment
- C08F8/14—Esterification
Definitions
- the present application relates to methods and compositions for liquid phase oligonucleotide synthesis employing the use of a polyvalent polymer hub.
- Oligonucleotide-based drugs have become a powerful epitome having ability to treat various diseases.
- the demand for oligonucleotides can be fulfilled by conventional solid phase oligonucleotide synthesis.
- Solid Phase Oligo Synthesis SPOS
- SPOS Solid Phase Oligo Synthesis
- the SPOS generally has low over all yield after multiple steps for an oligo sequence and high cost for reagents, solid support and waste management.
- SPOS may result in mismatched oligo sequences which leads to difficulty in purification.
- the increasing demand for metric ton quantities of oligonucleotides far exceeds the production capacity of solid phase oligonucleotide synthesis.
- Liquid phase oligonucleotide synthesis is a technology with the potential to provide the production capacity that will be required.
- One of the major advantages of LPOS over SPOS is the absence of the heterogeneous nature of the process, i.e., insoluble solid supports are not present.
- the use of a soluble scaffold or support employed in LPOS allows each step of the synthesis to be performed in the liquid phase.
- Some aspect of the present disclosure relates to a method for making oligonucleotides by liquid phase oligonucleotide synthesis (LPOS), comprising:
- PVH polyvalent hub
- the PVH comprises or is an acrylate polymer having a plurality of reactive ester groups capable of reacting with nucleoside or nucleotide analogs (i.e., anchor groups); and
- B 1 is a nitrogenous base
- G 1 is a 5′ hydroxyl blocking group
- R a is —H, —OH, halogen, —O—(C 1 -C 6 alkyl), —O—(C 1 -C 6 haloalkyl), or —OX, where X is a 2′ hydroxyl protecting group;
- n is an integer from 1 to about 500,000.
- the PVH used in the method comprises or is a homopolymer of the acrylate polymer.
- the PVH is a random copolymer of a reactive vinyl monomer (e.g., an acrylate monomer containing reactive ester functional group) and an acrylamide monomer.
- the formation of the first bioconjugate described in the method does not require a succinate linker to attach the nucleoside analog to the PVH.
- the method further comprises removing the 5′ blocking group (G 1 ) to form a 5′ unblocked first bioconjugate; and isolating the 5′ unblocked first bioconjugate.
- the 5′ unblocked first bioconjugate comprises the structure of Formula (I′):
- the method further comprises:
- Z is O or S
- Some other aspect of the present disclosure relates to oligonucleotides synthesized by the liquid phase oligo synthesis method described herein, wherein the oligonucleotides have at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 15 or 20 bases.
- PVH polyvalent hub
- R 1 is H or C 1 -C 6 alkyl
- R x is
- each R 2 is independently halogen, nitro or cyano; and n is 1, 2, 3, 4, or 5.
- the PVH may further comprise acrylamide repeating units or other acrylate repeating units that do not have reactive groups for attaching nucleoside or nucleotide analogs.
- the PVH comprises or is a copolymer, comprising one or more acrylate repeating units of Formula (II) or (II′) as described herein, or a combination thereof, and one or more acrylamide repeating units of Formula (III):
- R 3 is H or C 1 -C 6 alkyl; each of R 4 and R 5 is independently H, unsubstituted or substituted C 1 -C 6 alkyl, unsubstituted or substituted phenyl, or R 4 and R 5 together with the nitrogen atom to which they are attached form an optionally substituted 5 or 6 membered heterocyclyl.
- the number of the acrylate repeating unit is x
- the number of acrylamide repeating unit is y, where each x and y is independently an integer of 1 to 500,000.
- the copolymer comprises the structure:
- each of x and y is independently an integer from 1 to 500,000; and n is 4 or 5.
- Some additional aspect of the present disclosure relates to a polymeric bioconjugate for liquid phase oligonucleotide synthesis, said polymeric bioconjugate comprising one or more repeating units of Formula (V):
- each of R 1 and R 3 is independently H or C 1 -C 6 alkyl
- each of R 4 and R 5 is independently H, unsubstituted or substituted C 1 -C 6 alkyl, unsubstituted or substituted phenyl, or R 4 and R 5 together with the nitrogen atom to which they are attached form an optionally substituted 5 or 6 membered heterocyclyl;
- B 1 is a nitrogenous base
- G 1 is a 5′ hydroxyl blocking group
- R a is —H, —OH, halogen, —O—(C 1 -C 6 alkyl), —O—(C 1 -C 6 haloalkyl), or —OX, where X is a 2′ hydroxyl protecting group.
- the polymeric bioconjugate comprises or has the structure of Formula (Ia):
- each of x and m is independently an integer from 1 to about 500,000. In some further embodiments, m is less than y.
- Additional aspect of the present disclosure relate to a polymeric bioconjugate for liquid phase oligonucleotide synthesis, comprising one or more repeating units of Formula (VI):
- polymeric bioconjugate comprises or has the structure of Formula (Ib):
- each of x and m is independently an integer from 1 to about 500,000. In some further embodiments, m is equal to or less than y.
- the PVH or the PVH containing bioconjugate described herein may comprise or is a random copolymer having molecular weight ranging from about 10 kDa to about 1000 kDa, from about 20 KDa to about 500 KDa, or from about 30 kDa to about 100 kDa.
- FIG. 1 is a general reaction scheme for making an oligonucleotide by liquid phase oligonucleotide synthesis according to an embodiment of the present application.
- FIG. 2 is a reaction scheme of the reaction between 5′-O-DMT-dT and a polymer for liquid phase oligonucleotide synthesis followed by removal of the DMT group according to an embodiment of the present application.
- FIG. 3 A is a 1 H NMR spectrum measured in CD 3 CN of 5′-O-DMT-dT.
- FIG. 3 B is a 1 H NMR spectrum measured in CD 3 CN of a poly(TFPA-co-DMA) according to an embodiment of the present application.
- FIG. 3 C is a 1 H NMR spectrum measured in CD 3 CN of a DMT-dT conjugated poly(TFPA-co-DMA) according to an embodiment of the present application.
- FIG. 4 is a reaction scheme of the removal of DMT from a DMT-dT conjugated poly(TFPA-co-DMA) according to an embodiment of the present application.
- FIG. 5 is a reaction scheme of a first cycle of liquid phase oligonucleotide synthesis according to an embodiment of the present application.
- FIG. 6 is a reaction scheme for preparing a regenerated cellulose membrane for dialysis according to an embodiment of the present application.
- Solid phase oligonucleotide synthesis enable oligo synthesis at the solid support-liquid interface.
- the solid support is insoluble in the liquid medium (e.g., organic solvent).
- liquid medium e.g., organic solvent
- solid support include particles of controlled porous glass (CPG) and porous crosslink polystyrene.
- CPG controlled porous glass
- LPOS liquid phase oligo synthesis
- Conventional LPOS typically utilizes soluble supports that have one or several functional groups as anchors to conjugate and synthesize oligos.
- Embodiments of the present disclosure relate to methods for liquid phase oligonucleotide synthesis by using a polymeric soluble hub (i.e., polyvalent hub or PVH) that has numerous functional groups along the polymer chain for oligo synthesis.
- a polymeric soluble hub i.e., polyvalent hub or PVH
- the PVH described herein may contain reactive ester groups that allows for efficient conjugation with nucleoside or nucleotide analogs with improved yield compared to known liquid phase oligonucleotide synthesis and solid phase oligonucleotide synthesis.
- the PVH of the present disclosure eliminates the use of succinate linker to attach the nucleoside or nucleotide analogs to the PVH.
- the methods also utilize regenerated cellulose membrane to isolate and purify the growing oligo bioconjugates after each cycle of the synthesis.
- Regenerated cellulose membrane described herein is a cost-effective alternative to the nanofiltration technology used in the LPOS.
- the methods described herein is amenable for multi-kilogram oligonucleotide synthesis.
- the term “comprising” means that the compound, composition, or device includes at least the recited features or components, but may also include additional features or components.
- Consisting of is meant including, and limited to, whatever follows the phrase “consisting of.”
- Consisting essentially of is meant including any elements listed after the phrase, and limited to other elements that do not interfere with or contribute to the activity or action specified in the disclosure for the listed elements.
- average molecular weight is the weight-average molecular weight (Mw) of a sample population made up of polymer species having a multiplicity of molecular weights. This quantity is defined by the equation:
- n i indicates the number of molecules of species i and M i is the molecular weight of i th species.
- molecular weight refers to weight average molecular weight, unless otherwise specified.
- polymer used herein in its traditional sense, is a large molecule composed of smaller monomeric or oligomeric subunits covalently linked together to form a chain.
- a “homopolymer” is a polymer made up of only one monomeric repeating unit.
- a “copolymer” refers to a polymer made up of two or more kinds of monomeric repeating unit.
- Linear polymers are composed of monomeric subunits linked together in one continuous length to form polymer chains. Branched polymers are similar to linear polymers but have side chains protruding from various branch points along the main polymer.
- Star-shaped polymers are similar to branched polymers except that multiple side branches radiate from a single branch site, resulting in a star-shaped or wheel-and-spoke appearance.
- alkyl refers to a straight or branched hydrocarbon chain that is fully saturated (i.e., contains no double or triple bonds).
- the alkyl group may have 1 to 20 carbon atoms (whenever it appears herein, a numerical range such as “1 to 20” refers to each integer in the given range; e.g., “1 to 20 carbon atoms” means that the alkyl group may consist of 1 carbon atom, 2 carbon atoms, 3 carbon atoms, etc., up to and including 20 carbon atoms, although the present definition also covers the occurrence of the term “alkyl” where no numerical range is designated).
- the alkyl group may also be a medium size alkyl having 1 to 9 carbon atoms.
- the alkyl group could also be a lower alkyl having 1 to 6 carbon atoms.
- the alkyl group may be designated as “C 1-4 alkyl” or similar designations.
- C 1-6 alkyl indicates that there are one to six carbon atoms in the alkyl chain, i.e., the alkyl chain is selected from the group consisting of methyl, ethyl, propyl, iso-propyl, n-butyl, iso-butyl, sec-butyl, and t-butyl.
- Typical alkyl groups include, but are in no way limited to, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tertiary butyl, pentyl, hexyl, and the like.
- alkoxy refers to the formula —OR wherein R is an alkyl as is defined above, such as “C 1-9 alkoxy”, including but not limited to methoxy, ethoxy, n-propoxy, 1-methylethoxy (isopropoxy), n-butoxy, iso-butoxy, sec-butoxy, and tert-butoxy, and the like.
- heterocyclyl means a non-aromatic cyclic ring or ring system containing at least one heteroatom in the ring backbone. Heterocyclyls may be joined together in a fused, bridged or spiro-connected fashion. Heterocyclyls may have any degree of saturation provided that at least one ring in the ring system is not aromatic. The heteroatom(s) may be present in either a non-aromatic or aromatic ring in the ring system.
- the heterocyclyl group may have 3 to 20 ring members (i.e., the number of atoms making up the ring backbone, including carbon atoms and heteroatoms), although the present definition also covers the occurrence of the term “heterocyclyl” where no numerical range is designated.
- the heterocyclyl group may also be a medium size heterocyclyl having 3 to 10 ring members.
- the heterocyclyl group could also be a heterocyclyl having 3 to 6 ring members.
- the heterocyclyl group may be designated as “3-6 membered heterocyclyl” or similar designations.
- the heteroatom(s) are selected from one up to three of O, N or S, and in preferred five membered monocyclic heterocyclyls, the heteroatom(s) are selected from one or two heteroatoms selected from O, N, or S.
- heterocyclyl rings include, but are not limited to, azepinyl, acridinyl, carbazolyl, cinnolinyl, dioxolanyl, imidazolinyl, imidazolidinyl, morpholinyl, oxiranyl, oxepanyl, thiepanyl, piperidinyl, piperazinyl, dioxopiperazinyl, pyrrolidinyl, pyrrolidonyl, pyrrolidionyl, 4-piperidonyl, pyrazolinyl, pyrazolidinyl, 1,3-dioxinyl, 1,3-dioxanyl, 1,4-dioxinyl, 1,4-dioxanyl, 1,3-oxathianyl, 1,4-oxathiinyl, 1,4-oxathianyl, 2H-1,2-oxazinyl, trioxanyl, hexahydr
- cyano refers to a “—CN” group.
- a “nitro” group refers to a “—NO 2 ” group.
- a substituted group is derived from the unsubstituted parent group in which there has been an exchange of one or more hydrogen atoms for another atom or group.
- substituents independently selected from C 1 -C 6 alkyl, C 1 -C 6 alkenyl, C 1 -C 6 alkynyl, C 1 -C 6 heteroalkyl, C 3 -C 7 carbocyclyl (optionally substituted with halo, C 1 -C 6 alkyl, C 1 -C 6 alkoxy, C 1 -C 6 haloalkyl, and C 1 -C 6 haloalkoxy), C 3 -C 7 -carbocyclyl-C 1 -C 6 -alkyl (optionally substituted with halo, C 1 -C 6 alkyl, C 1 -C 6 alkoxy, C 1
- a “nucleotide” includes a nitrogen containing heterocyclic base, a sugar, and one or more phosphate groups. They are monomeric units of a nucleic acid sequence.
- the sugar is a ribose, and in DNA a deoxyribose, i.e. a sugar lacking a hydroxyl group that is present in ribose.
- the nitrogen containing heterocyclic base can be purine or pyrimidine base.
- Purine bases include adenine (A) and guanine (G), and modified derivatives or analogs thereof, such as deazapurine.
- Pyrimidine bases include cytosine (C), thymine (T), and uracil (U), and modified derivatives or analogs thereof.
- C cytosine
- T thymine
- U uracil
- the C-1 atom of deoxyribose is bonded to N-1 of a pyrimidine or N-9 of a purine.
- nucleoside is structurally similar to a nucleotide, but is missing the phosphate moieties.
- An example of a nucleoside analogue would be one in which the label is linked to the base and there is no phosphate group attached to the sugar molecule.
- nucleoside is used herein in its ordinary sense as understood by those skilled in the art. Examples include, but are not limited to, a ribonucleoside comprising a ribose moiety and a deoxyribonucleoside comprising a deoxyribose moiety.
- a modified pentose moiety is a pentose moiety in which an oxygen atom has been replaced with a carbon and/or a carbon has been replaced with a sulfur or an oxygen atom.
- a “nucleoside” is a monomer that can have a substituted base and/or sugar moiety. Additionally, a nucleoside can be incorporated into larger DNA and/or RNA polymers and oligomers.
- purine base is used herein in its ordinary sense as understood by those skilled in the art, and includes its tautomers.
- pyrimidine base is used herein in its ordinary sense as understood by those skilled in the art, and includes its tautomers.
- a non-limiting list of optionally substituted purine-bases includes purine, deazapurine, 7-deazapurine, adenine, 7-deaza adenine, guanine, 7-deaza guanine, hypoxanthine, xanthine, alloxanthine, 7-alkylguanine (e.g., 7-methylguanine), theobromine, caffeine, uric acid and isoguanine.
- pyrimidine bases include, but are not limited to, cytosine, thymine, uracil, 5,6-dihydrouracil and 5-alkylcytosine (e.g., 5-methylcytosine).
- “derivative” or “analogue” means a synthetic nucleoside or nucleotide derivative having modified base moieties and/or modified sugar moieties. Such derivatives and analogs are discussed in, e.g., Scheit, Nucleotide Analogs (John Wiley & Son, 1980) and Uhlman et al., Chemical Reviews 90:543-584, 1990. Nucleotide analogs can also comprise modified phosphodiester linkages, including phosphorothioate, phosphorodithioate, alkyl-phosphonate, phosphoranilidate and phosphoramidate linkages. “Derivative” and “analog” as used herein, may be used interchangeably, and are encompassed by the terms “nucleotide” and “nucleoside” defined herein.
- phosphate is used in its ordinary sense as understood by those skilled in the art, and includes its protonated forms (for example,
- protecting group and “blocking group” refer to any atom or group of atoms that is added to a molecule in order to prevent existing groups in the molecule from undergoing unwanted chemical reactions.
- Examples of protecting group moieties are described in T. W. Greene and P. G. M. Wuts, Protective Groups in Organic Synthesis, 3. Ed. John Wiley & Sons, 1999, and in J. F. W. McOmie, Protective Groups in Organic Chemistry Plenum Press, 1973, both of which are hereby incorporated by reference for the limited purpose of disclosing suitable protecting groups.
- the protecting group moiety may be chosen in such a way, that they are stable to certain reaction conditions and readily removed at a convenient stage using methodology known from the art.
- a non-limiting list of protecting groups include benzyl (Bn); substituted benzyl; alkylcarbonyls (e.g., t-butoxycarbonyl (BOC), acetyl (i.e., —C( ⁇ O)CH 3 or Ac), or isobutyryl (iBu); arylalkylcarbonyls (e.g., benzyloxycarbonyl or benzoyl (i.e., —C( ⁇ O)Ph or Bz)); substituted methyl ether (e.g., methoxymethyl ether (MOM)); substituted ethyl ether (e.g., methoxyethyl ether (MOE); a substituted benzyl ether; tetrahydropyranyl ether; silyl ethers (e.g., trimethylsilyl (TMS), triethylsilyl, triisopropylsilyl, t-butyldimethyl
- reactive ester refers to either an acyclic or a cyclic ester functional group comprising the moiety —C( ⁇ O)O— that is highly susceptible toward nucleophilic attack.
- loading capacity is expressed in mmol or ⁇ mol of a nucleoside bound to the polyvalent hub (PVH) described herein per gram of PVH (i.e., mmol/g).
- Some embodiments of the present application relate to a method for making a compound by liquid phase synthesis.
- the compound may be an oligonucleotide, a peptide, a polynucleotide (i.e., nucleic acid), or a small molecule.
- the method is for making an oligonucleotide by liquid phase oligonucleotide synthesis.
- the method is for making a peptide by liquid phase peptide synthesis.
- the method is for making a polynucleotide (i.e., nucleic acid) by liquid phase polynucleotide (i.e., nucleic acid) synthesis.
- the method is for making a small molecule by liquid phase small molecule synthesis.
- Embodiments of the method provided herein use a polyvalent hub (PVH) as alternative to a solid support for the synthesis.
- PVH polyvalent hub
- the method includes dissolving a polyvalent hub (PVH) in a first solvent to form a reaction matrix, contacting the PVH with one or more nucleoside analogs to form a first bioconjugate comprising a structure of Formula (I):
- PVH polyvalent hub
- B 1 is a nitrogenous base (such as purine, deazapurine or pyrimidine base); G 1 is a 5′ hydroxyl blocking group; R a is —H, —OH, halogen, —O—(C 1 -C 6 alkyl), —O—(C 1 -C 6 haloalkyl), or —OX, where X is a 2′ hydroxyl protecting group; and m is an integer from 1 to 500,000. In some such embodiments, m is from 4 to about 50,000. In further embodiments, the number of m is at least 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000.
- the structure of Formula (I) illustrates the attachment of the 3′ oxygen of the ribose ring of one or more nucleoside analogs to reactive sites of the PVH via an ester bond.
- B 1 is independently optionally protected adenine, optionally protected deaza adenine, optionally protected cytosine, optionally protected guanine, optionally protected deaza guanine, optionally protected thymine, or optionally protected uracil. In further embodiments, B 1 is
- R N is hydrogen, unsubstituted or substituted C 1 -C 6 alkyl, or an amino protecting group, or the hydrogen in —NHR N is absent and R N is a divalent amino protecting group.
- each G 1 is independently a trityl type of hydroxy protecting group selected from the group consisting of (4-methoxyphenyl)diphenylmethyl, bis(4-methoxyphenyl)phenylmethyl, tris(4-methoxyphenyl)methyl, 9-phenylxanthen-9-yl, and 9-(4-methoxyphenyl)xanthen-9-yl.
- each G 1 is independently a trityl type of hydroxy protecting group selected from the group consisting of (4-methoxyphenyl)diphenylmethyl, bis(4-methoxyphenyl)phenylmethyl, tris(4-methoxyphenyl)methyl, 9-phenylxanthen-9-yl, and 9-(4-methoxyphenyl)xanthen-9-yl.
- each G 1 is independently a trityl type of hydroxy protecting group selected from the group consisting of (4-methoxyphenyl)diphenylmethyl, bis(
- R b , R c and R d is independently H or C 1 -C 6 alkoxy.
- G 1 is bis(4-methoxyphenyl)phenylmethyl (4,4′-dimethoxytrityl).
- the PVH comprises or is an acrylate polymer having a plurality of reactive ester groups capable of reacting with nucleoside or nucleotide analogs.
- each reactive ester group of the PVH independently comprises —C( ⁇ O)OEW, NHS ester (e.g.,
- the PVH comprises —C( ⁇ O)—NH(CH 2 ) 2-5 OH or C( ⁇ O)—NH(CH 2 ) 2-5 NH 2 . In other embodiments, the PVH does not comprise —C( ⁇ O)—NH(CH 2 ) 2-5 OH or C( ⁇ O)—NH(CH 2 ) 2-5 NH 2 .
- the first bioconjugate described herein may include linkers between the reactive ester group the PVH and each of the nucleoside analogs.
- the linkers comprise C 2 -C 6 alkyl diamines, C 2 -C 6 hydroxy alkylamines, —C(O)O— groups and combinations thereof.
- the linkages comprise —C(O)O— groups.
- the linkages do not include a succinate or derivatives thereof.
- the formation of the first bioconjugate does not require a succinate linker to attach the nucleoside analog to the PVH.
- the acrylate polymer of the PVH comprises a plurality of repeating units of Formula (II) or (II′):
- R 1 is H or C 1 -C 6 alkyl
- R x is
- each R 2 is independently halogen, nitro or cyano; and n is 1, 2, 3, 4, or 5.
- R 1 is H or methyl.
- each R 2 is fluoro, and n is 3, 4, or 5.
- the PVH comprise
- R 1 is H, each R 2 is fluoro, and n is 4.
- the PVH comprises or is a copolymer comprising the acrylate polymer described herein and an acrylamide polymer.
- the acrylamide polymer of the PVH comprises comprising a plurality of repeating units of Formula (III):
- R 3 is H or C 1 -C 6 alkyl; and each of R 4 and R 5 is independently H, unsubstituted or substituted C 1 -C 6 alkyl, unsubstituted or substituted phenyl, or R 4 and R 5 together with the nitrogen atom to which they are attached form an optionally substituted 5 or 6 membered heterocyclyl.
- R 3 is H or methyl.
- each R 4 and R 5 is independently unsubstituted C 1 -C 6 alkyl.
- R 3 is H and each R 4 and R 5 is methyl.
- the molar ratio of the acrylate polymer to the acrylamide polymer in the copolymer is from about 1:200 to about 200:1, from about 1:100 to about 100:1, from about 1:90 to about 90:1, from about 1:80 to about 80:1, from about 1:70 to about 70:1, from about 1:60 to about 60:1, from about 1:50 to about 50:1, from about 1:20 to about 20:1, from about 1:10 to about 10:1, from about 5:1 to about 1:5; from about 2:1 to about 1:2; or about 1:1.
- the molar ratio of the acrylate polymer to the acrylamide polymer in the copolymer is about 1:5.
- the PVH comprises the structure:
- the ratio of x:y may range from about 1:1000 to about 1000:1, from about 1:500 to about 500:1, from about 1:200 to about 200:1, from about 1:100 to about 100:1, from about 1:90 to about 90:1, from about 1:80 to about 80:1, from about 1:70 to about 70:1, from about 1:60 to about 60:1, from about 1:50 to about 50:1, from about 1:20 to about 20:1, from about 1:10 to about 10:1, or from about 1:5 to about 5:1.
- the acrylate repeating units (y) in the copolymer is from about 0.1 mol % to about 100 mol %, form about 1 mol % to about 80 mol %, from about 5 mol % to about 60 mol %, or from about 10 mol % to about 50 mol %.
- the number of acrylate repeating units (i.e., y) in the PVH is at least 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000.
- PVH comprises or is
- the acrylate repeating units (y) in the copolymer is from about 0.1 mol % to about 100 mol %, form about 1 mol % to about 80 mol %, from about 5 mol % to about 60 mol %, or from about 10 mol % to about 50 mol %. In one embodiment, the acrylate repeating units is about 15 to 20 mol %, or about 18 mol %.
- the PVH has an average molecular weight from about 10 kDa to about 1000 kDa, or from about 20 kDa to about 500 kDa, or from about 30 kDa to about 100 kDa.
- the PVH having an average molecular weight (MW) of about 30 KDa or higher extends into the bulk of the liquid phase, resulting in improved reaction kinetics and reaction yield. Furthermore, such PVH also facilitates rapid discharge of reaction debris and unreacted biomolecules having an average MW of about 2 K or less.
- the first bioconjugate comprises the structure of Formula (Ia):
- the first bioconjugate comprises a random or block copolymer of
- each of R 1 and R 3 is H and each of R 4 and R 5 is methyl.
- the number of acrylate repeating units i.e., y is at least 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000.
- the method further comprising:
- the 5′ unblocked bioconjugate comprises the structure of Formula (Ia′):
- each of R 1 and R 3 is H and each of R 4 and R 5 is methyl.
- the isolation and/or purification of the 5′ unblocked first bioconjugate is achieved by a filtration step.
- the filtration step may include dialysis, filtration, nanofiltration, ultrafiltration, any known filtration technology suitable for use herein and combinations thereof.
- the filtration step comprises dialysis or filtration.
- filtration step includes the use of a membrane.
- the membrane may comprise a cellulose acetate, a glass fiber, a carbon-based polymer, a regenerated cellulose and combinations thereof.
- the regenerated cellulose membrane is negatively charged.
- the regenerated cellulose comprises the structure:
- the regenerated cellulose has a molecular weight cutoff (MWCO) from about 5 kDa to about 50 kDa, from about 6 kDa to about 40 kDa, about 7 kDa to about 30 kDa, or about 8 kDa to about 12 kDa.
- MWCO molecular weight cutoff
- the regenerated cellulose membrane is capable of retain the PVH containing bioconjugate as an alternative to the expensive nanofiltration membranes prepared with polyimide.
- the negatively charged membrane capable of reducing non-specific adsorption of negatively charged biomolecules.
- the regenerated cellulose is treated in a process including carbon disulfide followed by an aqueous metal hydroxide.
- the regenerated cellulose comprises dithioate groups and metal cations.
- the metal cations comprise group 1 metals (i.e., group IA metals or alkali metals), group 2 metals (i.e., group IIA metals or alkaline earth metals) and combinations thereof.
- the metal cations comprise sodium cations.
- the regenerated cellulose has an electrostatic charge.
- the method further comprising:
- G 2 is a 5′ hydroxyl blocking group
- B 2 is a nitrogenous base (e.g., purine, deazapurine, or pyrimidine base);
- R e is a phosphite protecting group; and
- p is an integer of 1 to 500,000;
- Z is O or S
- B 2 is independently optionally protected adenine, optionally protected deaza adenine, optionally protected cytosine, optionally protected guanine, optionally protected deaza guanine, optionally protected thymine, or optionally protected uracil. In further embodiments, B 2 is
- each G 2 is independently a trityl type of hydroxy protecting group selected from the group consisting of (4-methoxyphenyl)diphenylmethyl, bis(4-methoxyphenyl)phenylmethyl, tris(4-methoxyphenyl)methyl, 9-phenylxanthen-9-yl, and 9-(4-methoxyphenyl)xanthen-9-yl.
- each G 2 is independently a trityl type of hydroxy protecting group selected from the group consisting of (4-methoxyphenyl)diphenylmethyl, bis(4-methoxyphenyl)phenylmethyl, tris(4-methoxyphenyl)methyl, 9-phenylxanthen-9-yl, and 9-(4-methoxyphenyl)xanthen-9-yl.
- each G 2 is independently
- each of R b , R c and R d is independently H or C 1 -C 6 alkoxy.
- G 2 is bis(4-methoxyphenyl)phenylmethyl (4,4′-dimethoxytrityl).
- R e is an unsubstituted or substituted C 1 -C 6 alkyl.
- R e is 2-cyanoethyl (—CH 2 CH 2 CN).
- p is from 4 to about 50,000.
- the method further comprises blocking unreacted 5′ hydroxyl group in the 5′ unblocked first bioconjugate prior to step (b).
- said blocking is performed by reacting the 5′ hydroxyl group with acetic anhydride (Ac 2 O).
- the isolation and/or purification of the 5′ unblocked second bioconjugate is achieved by a filtration or dialysis described herein.
- the filtration may use a regenerated cellulose membrane described herein.
- the regenerated cellulose membrane has a molecular weight cutoff (MWCO) from about 5 kDa to about 50 kDa, from about 6 kDa to about 40 kDa, about 7 kDa to about 30 kDa, or about 8 kDa to about 12 kDa.
- steps (a)-(d) are repeated multiple cycles until a desired length of oligonucleotide has been synthesized.
- the oligonucleotide synthesized may comprises at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 bases.
- the method further comprises removing the synthesized oligonucleotides from the PVH.
- the removing step includes a step of covalent chemical bond scission.
- the removing step includes hydrolysis.
- the removing includes hydrolysis at a temperature from about 0° C. to about 80° C., or about 10° C. to about 60° C., or about 15° C. to about 30° C.
- FIG. 1 A general reaction scheme of the liquid phase oligonucleotide synthesis is illustrated in FIG. 1 .
- a more detailed reaction scheme according to an embodiment of the method described herein is illustrated in FIG. 5 .
- the nucleoside analog has methoxy or fluoro substitution at the 2′ position of the ribose ring.
- the same method may also be used for other types of nucleoside analogs where the corresponding R may be H (i.e., 2-deoxy ribose), or —OR a as described herein.
- FIG. 6 A reaction scheme for preparing the regenerated cellulose is described in FIG. 6 .
- each of the first solvent and the second solvent comprise one or more non-protic polar solvents, or combinations thereof.
- the one or more non-protic polar solvents comprise acetonitrile, tetrahydrofuran (THF), dimethylformamide (DMF), dimethyl sulfoxide (DMSO), dichloromethane (DCM), sulfolane, or combinations thereof.
- the first solvent and/or the second solvent comprises acetonitrile.
- the first solvent and/or the second solvent comprises a mixture of acetonitrile and sulfolane.
- the liquid phase synthesis comprises liquid phase oligonucleotide synthesis, liquid phase peptide synthesis, liquid phase polynucleotide (i.e., nucleic acid), synthesis or liquid phase small molecule synthesis.
- the PVH comprises or is a polymer for liquid phase oligonucleotide synthesis.
- the PVH is prepared from one or more monomers to produce the PVH having one or more repeating units.
- the monomers may include acrylic or methacrylic acid esters (e.g., acrylate) or combinations thereof, where certain acrylate monomers each contains a reactive ester group that allows for reaction with nucleoside or nucleotide analogs.
- the PVH may also contains repeating units of acrylic or methacrylic acid amides (e.g., acrylamide), or combination thereof.
- the PVH may comprise a linear polymer, a branched polymer, or star-shaped polymer, or combinations thereof.
- the PVH may comprise a homopolymer, a block copolymer, or a random copolymer, or combinations thereof.
- the PVH comprises a repeating unit of Formula (II) or (II′):
- R 1 is H or C 1 -C 6 alkyl
- R x is
- each R 2 is independently halogen, nitro or cyano; n is 1, 2, 3, 4, or 5; and wherein —OR x in Formula (II) is a leaving group for attaching a 3′-oxygen of a nucleoside or nucleotide analog to the carbonyl group.
- R 1 is methyl, ethyl, propyl, butyl, pentyl or hexyl.
- R 1 is H.
- each R 2 is fluoro, chloro, nitro or cyano and n is 3, 4, or 5.
- each R 2 is fluoro or chloro, and n is 3, 4, or 5.
- each R 2 is fluoro and n is 3, 4, or 5. In some embodiments, each R 2 is chloro and n is 3, 4, or 5. In some embodiments, each R 2 is nitro and n is 3, 4, or 5. In some embodiments, each R 2 is cyano and n is 3, 4, or 5. In some embodiments, each R 2 is fluoro and n is 4. In some embodiments, each R 2 is fluoro and n is 5.
- the PVH comprises a second repeating unit of Formula (III):
- R 3 is H or C 1 -C 6 alkyl; and each of R 4 and R 5 is independently H, unsubstituted or substituted C 1 -C 6 alkyl, unsubstituted or substituted phenyl, or R 4 and R 5 together with the nitrogen atom to which they are attached form an optionally substituted 5 or 6 membered heterocyclyl.
- R 3 is methyl, ethyl, propyl, butyl, pentyl or hexyl.
- R 3 is H.
- each R 4 and R 5 is independently unsubstituted C 1 -C 6 alkyl.
- each R 4 and R 5 is methyl.
- R 4 and R 5 together with the nitrogen atom to which they are attached form a structure selected from
- the PVH comprises or is a copolymer, comprising one or more acrylate repeating units of Formula (II) or (II′), or a combination thereof, and one or more acrylamide repeating units of Formula (III) as described herein, comprising or having the structure:
- the copolymer may be a block copolymer or a random copolymer:
- R 1 is H.
- each of R 4 and R 5 is methyl.
- the PVH comprises or has the structure:
- x is an integer from 1 to 500,000; y is an integer from 1 to 500,000; and n is 4 or 5. In one embodiment, n is 4. In some further embodiments, x is an integer from 20 to 10,000 and y is an integer from 4 to 2,000.
- the ratio of x:y may range from about 1:1000 to about 1000 to 1, from about 1:500 to about 500:1, from about 1:200 to about 200:1, from about 1:100 to about 100:1, from about 1:90 to about 90:1, from about 1:80 to about 80:1, from about 1:70 to about 70:1, from about 1:60 to about 60:1, from about 1:50 to about 50:1, from about 1:20 to about 20:1, from about 1:10 to about 10:1, or from about 1:5 to about 5:1.
- the acrylate repeating units (y) in the copolymer is from about 0.1 mol % to about 100 mol %, form about 1 mol % to about 80 mol %, from about 5 mol % to about 60 mol %, or from about 10 mol % to about 50 mol %.
- the number of acrylate repeating units (i.e., y) in the PVH is at least 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000.
- the PVH comprises 2,3,5,6-tetrafluorophenyl acrylate (TFPA) and N,N-dimethyl acrylamide (DMA) wherein the PVH can be referred to with the abbreviation poly(TFPA-co-DMA), having the structure
- the acrylate repeating units (y) in the copolymer is from about 0.1 mol % to about 100 mol %, form about 1 mol % to about 80 mol %, from about 5 mol % to about 60 mol %, or from about 10 mol % to about 50 mol %. In one embodiment, the acrylate repeating units is about 15 to 20 mol %, or about 18 mol %.
- the PVH has an average molecular weight from about 10 kDa to about 1000 kDa, or from about 20 kDa to about 500 kDa, or from about 30 kDa to about 100 kDa.
- the PVH may have a loading capacity of from about 10 ⁇ mol to about 600 ⁇ mol, from about 20 ⁇ mol to about 500 ⁇ mol, from about 30 ⁇ mol to about 400 ⁇ mol, or from about 40 ⁇ mol to about 300 ⁇ mol, per gram of PVH.
- the PVH has a loading capacity of about 10 ⁇ mol, 20 ⁇ mol, 30 ⁇ mol, 40 ⁇ mol, 50 ⁇ mol, 60 ⁇ mol, 70 ⁇ mol, 80 ⁇ mol, 90 ⁇ mol, 100 ⁇ mol, 150 ⁇ mol, 200 ⁇ mol, 250 ⁇ mol, 300 ⁇ mol, 350 ⁇ mol, 400 ⁇ mol, 450 ⁇ mol, 500 ⁇ mol, 550 ⁇ mol or 600 ⁇ mol per gram of PVH, or a range defined by any two of the preceding values.
- the PVH may be used in the liquid phase synthesis method described herein.
- Some additional aspect of the present disclosure relates to a polymeric bioconjugate comprising one or more repeating units of Formula (V):
- each of R 1 and R 3 is independently H or C 1 -C 6 alkyl
- each of R 4 and R 5 is independently H, unsubstituted or substituted C 1 -C 6 alkyl, unsubstituted or substituted phenyl, or R 4 and R 5 together with the nitrogen atom to which they are attached form an optionally substituted 5 or 6 membered heterocyclyl;
- B 1 is a nitrogenous base described herein;
- G 1 is H or a 5′ hydroxyl blocking group
- R a is —H, —OH, halogen, —O—(C 1 -C 6 alkyl), —O—(C 1 -C 6 haloalkyl), or —OX, where X is a 2′ hydroxyl protecting group.
- the polymeric bioconjugate comprises or has the structure of Formula (Ia):
- each of x, and m is independently an integer from 1 to 500,000. In some embodiments, m is equal to less than y as described herein.
- each of R 1 and R 3 is H. In further embodiments, each of R 4 and R 5 is methyl. In further embodiments, G 1 is H. In other embodiments, G 1 is
- each of R b , R c and R d is independently H or C 1 -C 6 alkoxy.
- G 1 is 4,4′-dimethoxytrityl.
- polymeric bioconjugate comprising one or more repeating units of Formula (VI), and optionally one or more repeating unit of Formula (III):
- polymeric bioconjugate comprises or has the structure of Formula (Ib):
- each of x, and m is independently an integer from 1 to 500,000. In some embodiments, m is equal to less than y as described herein.
- each of R 1 and R 3 is H. In further embodiments, each of R 4 and R 5 is methyl.
- G 2 is H. In other embodiments, G 2 is
- each of R b , R c and R d is independently H or C 1 -C 6 alkoxy.
- G 2 is 4,4′-dimethoxytrityl.
- R e is substituted C 1 -C 6 alkyl.
- R e is 2-cyanoethyl (—CH 2 CH 2 CN).
- B 2 is independently optionally protected adenine, optionally protected deaza adenine, optionally protected cytosine, optionally protected guanine, optionally protected deaza guanine, optionally protected thymine, or optionally protected uracil. In further embodiments, B 2 is
- R N is hydrogen, unsubstituted or substituted C 1 -C 6 alkyl, or an amino protecting group, or the hydrogen in —NHR N is absent and R N is a divalent amino protecting group.
- reaction mixture was cooled to ambient temperature in open air.
- the AcN and residual monomers were removed under reduced pressure in a Rota-Vap at 55° C. water-bath temperature.
- the polymer product was re-dissolved in 30 mL of anhydrous tetrahydrofuran (THF) at 55° C.
- THF anhydrous tetrahydrofuran
- the cloudy suspension was then added into 800 mL of anhydrous hexane in a 2-L Erlenmeyer flask through a 22-gauge syringe needle in a fine stream under argon while mechanically stirred at about 150 rpm with a 2-inch Teflon stirring blade.
- the precipitated polymer was stirred for an additional 5 minutes under argon.
- the hexane was discarded, and 500 mL of fresh anhydrous hexane was added and mechanically stirred gently for another 15 minutes under argon.
- the poly(TFPA-co-DMA) product in coarse fibers was then transferred into a large mouth 500-mL glass jar and dried under vacuum at 55° C. for 24 hours.
- the TFPA proportion in the poly(TFPA-co-DMA) product was determined to be 18 mol % by 1 H NMR analysis.
- a solution of 312.8 mg of poly(TFPA-co-DMA) prepared in Example 1 and 10.0 mL of anhydrous AcN was prepared and transferred into a dialysis tube having a length of 10 cm.
- the dialysis tube (Spectra/Por-7 Dialysis membrane, 15 KDa MWCO, 2.4 cm flat width, 1.5 cm diameter, and 1.8 mL/cm capacity) was immersed into a dialysis bath containing 200.0 mL AcN as a dialysis solvent. Dialysis was conducted for 24 h and afterward the dialysis tube was separated from the dialysis bath.
- the solution in the dialysis tube was transferred to a recovery flask and the dialysis tube was rinsed with AcN.
- the solution and rinsings were combined and the AcN in the recovery flask was removed by a Rota-Vap at 50 mbar.
- the residue was subjected to vacuum drying and 282.70 mg of poly(TFPA-co-DMA) (90.4%) was observed in the dialysis tube as shown in Table 1.
- Dialysis using a membrane having an MWCO of 15 KDa retained more than 90% poly(TFPA-co-DMA) in the dialysis tube.
- the dialysis solvent was transferred into a recovery flask and the dialysis bath was rinsed with AcN.
- the dialysis solvent and rinsings were combined and the AcN in the recovery flask was removed by a Rota-Vap at 50 mbar.
- the recovery flask was rinsed with two 10 mL portions of water and the water-clear rinsings were concentrated under vacuum to recover 1.50 mg of the PVH copolymer as shown in Table 1.
- the dialysis tube 18 cm in length (15 KDa MWCO, 2.4 cm flat width, 1.5 cm diameter, and 1.8 mL/cm capacity) and immersed into 400 mL of AcN.
- the dialysis was allowed to proceed for 23 hours with one change of 400 mL can (a total of 800 mL of AcN).
- the reaction solution in the dialysis tube was transferred into a 100-mL round bottom flask.
- the dialysis tube was rinsed with two 10-mL aliquots of can and the rinsings were added into the 100-mL flask.
- FIG. 2 illustrates the steps of (i) reaction between 5′-O-DMT-dT and poly(TFPA-co-DMA), (ii) a first dialysis; (iii) removal of the DMT group; and (iv) a second dialysis.
- FIGS. 3 A, 3 B and 3 C illustrate 1 H NMR spectra taken in AcN-d3 for 5′-O-DMT-dT, the poly(TFPA-co-DMA) prepared in Example 1 and the DMT-dT conjugated poly(TFPA-co-DMA) prepared in Example 4, respectively. Comparison of the 1 H NMR integrations of various signals indicates that 5′-O-DMT-dT had been conjugated on the PVH.
- the maximum amount of free 5′-O-DMT-dT in a 15-mg sample of the DMT-dT conjugated poly(TFPA-co-DMA) prepared for NMR spectroscopy is 0.007 mg, which under the detection limit of NMR instrumentation.
- DMT-dT loading of the DMT-dT conjugated poly(TFPA-co-DMA) was determined by UV/Vis spectrophotometry.
- the DMT group is removed from dT under acidic hydrolysis as shown in FIG. 4 and the loading of DMT-dT was quantitatively determined.
- An aliquot of 2.20 mg of the DMT-dT conjugated poly(TFPA-co-DMA) prepared in Example 4 was washed with 100 mL of AcN containing 0.46 M toluenesulfonic acid (TSA-AcN).
- TSA-AcN 0.46 M toluenesulfonic acid
- a blank UV/Vis spectrum of 0.46 M TSA-AcN solution was acquired.
- An aliquot of the supernatant from the washing solution was transferred to a dry cuvette and UV/VIS scanning was performed.
- the DMT-dT loading on the DMT-dT conjugated poly(TFPA-co-DMA) was determined from an absorbance value (A) of 0.59674 at 498 nm with a sample volume of 100 mL.
- the extinction coefficient ⁇ for DMT was estimated 76.5 mL/cm* ⁇ mole.
- DMT-dT loading ( ⁇ mole/g) [A*sample volume (mL)]/[76.5 (mL*cm ⁇ 1 * ⁇ mole ⁇ 1 )*sample weight (g)]
- DMT-dT loading of the DMT-dT conjugated poly(TFPA-co-DMA) was calculated to be 355 ⁇ mol/g.
Landscapes
- Chemical & Material Sciences (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- Medicinal Chemistry (AREA)
- Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Biochemistry (AREA)
- Molecular Biology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Polymers & Plastics (AREA)
- Pharmacology & Pharmacy (AREA)
- Epidemiology (AREA)
- Animal Behavior & Ethology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Biotechnology (AREA)
- Genetics & Genomics (AREA)
- General Chemical & Material Sciences (AREA)
- Saccharide Compounds (AREA)
Abstract
Description
-
- (a) reacting the 5′ unblocked first bioconjugate with one or more nucleoside phosphoramidite analogs in a second solvent to form a second bioconjugate comprising the structure of Formula (IV):
-
- G2 is a 5′ hydroxyl blocking group;
- B2 is a nitrogenous base;
- Re is a phosphite protecting group; and
- p is an integer of 1 to about 500,000;
- (b) oxidizing the phosphite moiety in Formula (IV);
- (c) removing the 5′ blocking group G2 to form a 5′ unblocked second bioconjugate comprising the structure of Formula (IV′):
-
- (d) isolating the 5′ unblocked second bioconjugate. In some embodiments, steps (a)-(d) are repeated multiple cycles until a desired length of oligonucleotide has been synthesized.
each R2 is independently halogen, nitro or cyano; and n is 1, 2, 3, 4, or 5. In some embodiments, the PVH may further comprise acrylamide repeating units or other acrylate repeating units that do not have reactive groups for attaching nucleoside or nucleotide analogs. In some further embodiment, the PVH comprises or is a copolymer, comprising one or more acrylate repeating units of Formula (II) or (II′) as described herein, or a combination thereof, and one or more acrylamide repeating units of Formula (III):
wherein R3 is H or C1-C6 alkyl; each of R4 and R5 is independently H, unsubstituted or substituted C1-C6 alkyl, unsubstituted or substituted phenyl, or R4 and R5 together with the nitrogen atom to which they are attached form an optionally substituted 5 or 6 membered heterocyclyl. In some embodiments, the number of the acrylate repeating unit is x, and the number of acrylamide repeating unit is y, where each x and y is independently an integer of 1 to 500,000.
wherein each of x and m is independently an integer from 1 to about 500,000. In some further embodiments, m is less than y.
-
- each of R1 and R3 is independently H or C1-C6 alkyl;
- each of R4 and R5 is independently H, unsubstituted or substituted C1-C6 alkyl, unsubstituted or substituted phenyl, or R4 and R5 together with the nitrogen atom to which they are attached form an optionally substituted 5 or 6 membered heterocyclyl;
- Ra is —H, —OH, halogen, —O—(C1-C6 alkyl), —O—(C1-C6 haloalkyl), or —OX, where X is a 2′ hydroxyl protecting group.
- each of B1 and B2 is independently a nitrogenous base;
- G2 is a 5′ hydroxyl blocking group;
- Re is unsubstituted or substituted C1-C6 alkyl;
- Z is O or S; and
- k is an integer from 1 to 500.
wherein each of x and m is independently an integer from 1 to about 500,000. In some further embodiments, m is equal to or less than y.
As used herein, the terms “monophosphate,” “diphosphate,” and “triphosphate” are used in their ordinary sense as understood by those skilled in the art, and include protonated forms.
wherein B1 is a nitrogenous base (such as purine, deazapurine or pyrimidine base); G1 is a 5′ hydroxyl blocking group; Ra is —H, —OH, halogen, —O—(C1-C6 alkyl), —O—(C1-C6 haloalkyl), or —OX, where X is a 2′ hydroxyl protecting group; and m is an integer from 1 to 500,000. In some such embodiments, m is from 4 to about 50,000. In further embodiments, the number of m is at least 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000. The structure of Formula (I) illustrates the attachment of the 3′ oxygen of the ribose ring of one or more nucleoside analogs to reactive sites of the PVH via an ester bond.
wherein RN is hydrogen, unsubstituted or substituted C1-C6 alkyl, or an amino protecting group, or the hydrogen in —NHRN is absent and RN is a divalent amino protecting group.
wherein each of Rb, Rc and Rd is independently H or C1-C6 alkoxy. In one embodiment, G1 is bis(4-methoxyphenyl)phenylmethyl (4,4′-dimethoxytrityl).
wherein EW is an electron-withdrawing group. In some embodiments, the PVH comprises —C(═O)—NH(CH2)2-5OH or C(═O)—NH(CH2)2-5NH2. In other embodiments, the PVH does not comprise —C(═O)—NH(CH2)2-5OH or C(═O)—NH(CH2)2-5NH2.
each R2 is independently halogen, nitro or cyano; and n is 1, 2, 3, 4, or 5. In some such embodiments, R1 is H or methyl. In some further embodiments, each R2 is fluoro, and n is 3, 4, or 5. In one embodiment, the PVH comprise
wherein R3 is H or C1-C6 alkyl; and each of R4 and R5 is independently H, unsubstituted or substituted C1-C6 alkyl, unsubstituted or substituted phenyl, or R4 and R5 together with the nitrogen atom to which they are attached form an optionally substituted 5 or 6 membered heterocyclyl. In some such embodiments, R3 is H or methyl. In some such embodiments, each R4 and R5 is independently unsubstituted C1-C6 alkyl. In one embodiment, R3 is H and each R4 and R5 is methyl. In some embodiments, the molar ratio of the acrylate polymer to the acrylamide polymer in the copolymer is from about 1:200 to about 200:1, from about 1:100 to about 100:1, from about 1:90 to about 90:1, from about 1:80 to about 80:1, from about 1:70 to about 70:1, from about 1:60 to about 60:1, from about 1:50 to about 50:1, from about 1:20 to about 20:1, from about 1:10 to about 10:1, from about 5:1 to about 1:5; from about 2:1 to about 1:2; or about 1:1. In one embodiment, the molar ratio of the acrylate polymer to the acrylamide polymer in the copolymer is about 1:5. In some further embodiments, the PVH comprises the structure:
wherein x is an integer from 1 to 500,000; y is an integer from 1 to 500,000; and n is 4 or 5. The ratio of x:y may range from about 1:1000 to about 1000:1, from about 1:500 to about 500:1, from about 1:200 to about 200:1, from about 1:100 to about 100:1, from about 1:90 to about 90:1, from about 1:80 to about 80:1, from about 1:70 to about 70:1, from about 1:60 to about 60:1, from about 1:50 to about 50:1, from about 1:20 to about 20:1, from about 1:10 to about 10:1, or from about 1:5 to about 5:1. In some embodiments, the acrylate repeating units (y) in the copolymer is from about 0.1 mol % to about 100 mol %, form about 1 mol % to about 80 mol %, from about 5 mol % to about 60 mol %, or from about 10 mol % to about 50 mol %. In further embodiments, the number of acrylate repeating units (i.e., y) in the PVH is at least 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000.
and n is 4, such copolymer of 2,3,5,6-tetrafluorophenyl acrylate (TFPA) and N,N-dimethyl acrylamide (DMA) can be referred to with the abbreviation poly(TFPA-co-DMA). In some embodiments, the acrylate repeating units (y) in the copolymer is from about 0.1 mol % to about 100 mol %, form about 1 mol % to about 80 mol %, from about 5 mol % to about 60 mol %, or from about 10 mol % to about 50 mol %. In one embodiment, the acrylate repeating units is about 15 to 20 mol %, or about 18 mol %.
wherein x is an integer from 1 to 500,000; and m is an integer from 1 to 500,000. The first bioconjugate comprises a random or block copolymer of
where m is equal or less than y as described herein. In some further embodiments, each of R1 and R3 is H and each of R4 and R5 is methyl. In further embodiments, the number of acrylate repeating units (i.e., y) is at least 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000.
In some such embodiments, wherein the 5′ unblocked bioconjugate comprises the structure of Formula (Ia′):
wherein G2 is a 5′ hydroxyl blocking group; B2 is a nitrogenous base (e.g., purine, deazapurine, or pyrimidine base); Re is a phosphite protecting group; and p is an integer of 1 to 500,000;
wherein RN is hydrogen, unsubstituted or substituted C1-C6 alkyl, or an amino protecting group, or the hydrogen in —NHRN is absent and RN is a divalent amino protecting group. In some embodiments, each G2 is independently a trityl type of hydroxy protecting group selected from the group consisting of (4-methoxyphenyl)diphenylmethyl, bis(4-methoxyphenyl)phenylmethyl, tris(4-methoxyphenyl)methyl, 9-phenylxanthen-9-yl, and 9-(4-methoxyphenyl)xanthen-9-yl. In some further embodiments, each G2 is independently
wherein each of Rb, Rc and Rd is independently H or C1-C6 alkoxy. In one embodiment, G2 is bis(4-methoxyphenyl)phenylmethyl (4,4′-dimethoxytrityl). In some such embodiments, Re is an unsubstituted or substituted C1-C6 alkyl. In one example, Re is 2-cyanoethyl (—CH2CH2CN). In some such embodiments, p is from 4 to about 50,000.
each R2 is independently halogen, nitro or cyano; n is 1, 2, 3, 4, or 5; and wherein —ORx in Formula (II) is a leaving group for attaching a 3′-oxygen of a nucleoside or nucleotide analog to the carbonyl group. In some embodiments, R1 is methyl, ethyl, propyl, butyl, pentyl or hexyl. In some embodiments, R1 is H. In some embodiments, each R2 is fluoro, chloro, nitro or cyano and n is 3, 4, or 5. In some embodiments, each R2 is fluoro or chloro, and n is 3, 4, or 5. In some embodiments, each R2 is fluoro and n is 3, 4, or 5. In some embodiments, each R2 is chloro and n is 3, 4, or 5. In some embodiments, each R2 is nitro and n is 3, 4, or 5. In some embodiments, each R2 is cyano and n is 3, 4, or 5. In some embodiments, each R2 is fluoro and n is 4. In some embodiments, each R2 is fluoro and n is 5.
wherein R3 is H or C1-C6 alkyl; and each of R4 and R5 is independently H, unsubstituted or substituted C1-C6 alkyl, unsubstituted or substituted phenyl, or R4 and R5 together with the nitrogen atom to which they are attached form an optionally substituted 5 or 6 membered heterocyclyl. In some embodiments, R3 is methyl, ethyl, propyl, butyl, pentyl or hexyl. In some embodiments, R3 is H. In some embodiments, each R4 and R5 is independently unsubstituted C1-C6 alkyl. In some embodiments, each R4 and R5 is methyl. In some embodiments, R4 and R5 together with the nitrogen atom to which they are attached form a structure selected from
The copolymer may be a block copolymer or a random copolymer: In some embodiments, R1 is H. In further embodiments, each of R4 and R5 is methyl. In some further embodiments, the PVH comprises or has the structure:
wherein x is an integer from 1 to 500,000; y is an integer from 1 to 500,000; and n is 4 or 5. In one embodiment, n is 4. In some further embodiments, x is an integer from 20 to 10,000 and y is an integer from 4 to 2,000. The ratio of x:y may range from about 1:1000 to about 1000 to 1, from about 1:500 to about 500:1, from about 1:200 to about 200:1, from about 1:100 to about 100:1, from about 1:90 to about 90:1, from about 1:80 to about 80:1, from about 1:70 to about 70:1, from about 1:60 to about 60:1, from about 1:50 to about 50:1, from about 1:20 to about 20:1, from about 1:10 to about 10:1, or from about 1:5 to about 5:1. In some embodiments, the acrylate repeating units (y) in the copolymer is from about 0.1 mol % to about 100 mol %, form about 1 mol % to about 80 mol %, from about 5 mol % to about 60 mol %, or from about 10 mol % to about 50 mol %. In further embodiments, the number of acrylate repeating units (i.e., y) in the PVH is at least 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000.
where n is 4. In some embodiments, the acrylate repeating units (y) in the copolymer is from about 0.1 mol % to about 100 mol %, form about 1 mol % to about 80 mol %, from about 5 mol % to about 60 mol %, or from about 10 mol % to about 50 mol %. In one embodiment, the acrylate repeating units is about 15 to 20 mol %, or about 18 mol %.
wherein each of x, and m is independently an integer from 1 to 500,000. In some embodiments, m is equal to less than y as described herein. In further embodiments, each of R1 and R3 is H. In further embodiments, each of R4 and R5 is methyl. In further embodiments, G1 is H. In other embodiments, G1 is
wherein each of Rb, Rc and Rd is independently H or C1-C6 alkoxy. In one embodiment, G1 is 4,4′-dimethoxytrityl.
-
- each R1 of R3 is independently H or C1-C6 alkyl;
- each of R4 and R5 is independently H, unsubstituted or substituted C1-C6 alkyl, unsubstituted or substituted phenyl, or R4 and R5 together with the nitrogen atom to which they are attached form an optionally substituted 5 or 6 membered heterocyclyl;
- Ra is —H, —OH, halogen, —O—(C1-C6 alkyl), —O—(C1-C6 haloalkyl), or —OX, where X is a 2′ hydroxyl protecting group.
- each of B1 and B2 is independently a nitrogenous base described herein;
- G2 is a 5′ hydroxyl blocking group;
- Re is unsubstituted or substituted C1-C6 alkyl;
- Z is O or S; and
- k is an integer from 1 to 500.
wherein each of x, and m is independently an integer from 1 to 500,000. In some embodiments, m is equal to less than y as described herein. In further embodiments, each of R1 and R3 is H. In further embodiments, each of R4 and R5 is methyl. In further embodiments, G2 is H. In other embodiments, G2 is
wherein each of Rb, Rc and Rd is independently H or C1-C6 alkoxy. In one embodiment, G2 is 4,4′-dimethoxytrityl. In some embodiments, Re is substituted C1-C6 alkyl. In one example, Re is 2-cyanoethyl (—CH2CH2CN).
wherein RN is hydrogen, unsubstituted or substituted C1-C6 alkyl, or an amino protecting group, or the hydrogen in —NHRN is absent and RN is a divalent amino protecting group.
TABLE 1 | ||||||
5′-O- | Recovery | Recovery | dT | |||
poly(TFPA- | DMT- | from | from | Polymer | ex- | |
Exam- | co-DMA) | dT | dialysis | dialysis | retained | cluded |
ple | (mg) | (mg) | bath (mg) | tube (mg) | (%) | (%) |
2 | 312.8 | — | 1.50 | 282.70 | 90.4 | — |
3 | — | 218.7 | 196.70 | 10.10 | — | 90.0 |
4 | 320.0 | 226.2 | Not | 284.90 | Not | Not |
deter- | deter- | deter- | ||||
mined | mined | mined | ||||
Claims (31)
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US17/562,714 US11725073B2 (en) | 2020-12-29 | 2021-12-27 | Compositions and methods for liquid phase oligonucleotide synthesis |
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US202063131449P | 2020-12-29 | 2020-12-29 | |
US17/562,714 US11725073B2 (en) | 2020-12-29 | 2021-12-27 | Compositions and methods for liquid phase oligonucleotide synthesis |
Publications (2)
Publication Number | Publication Date |
---|---|
US20220204670A1 US20220204670A1 (en) | 2022-06-30 |
US11725073B2 true US11725073B2 (en) | 2023-08-15 |
Family
ID=82118908
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
US17/562,714 Active US11725073B2 (en) | 2020-12-29 | 2021-12-27 | Compositions and methods for liquid phase oligonucleotide synthesis |
Country Status (1)
Country | Link |
---|---|
US (1) | US11725073B2 (en) |
Citations (14)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
FR2623510A1 (en) | 1987-11-20 | 1989-05-26 | Ecole Nale Superieure Chimie | Polymers based on pentafluorophenyl halo- and methacrylates and their application to the production of optical fibres, of positive photoresists, of organic glasses and of resins for optical discs |
EP0850949A2 (en) | 1993-03-26 | 1998-07-01 | Institut Pasteur | Novel derivatives for use in nucleic acid sequencing |
WO2002079215A1 (en) | 2001-03-30 | 2002-10-10 | Isis Pharmaceuticals, Inc. | Building blocks for the solution phase synthesis of oligonucleotides |
WO2003093346A1 (en) | 2002-05-06 | 2003-11-13 | Universita' Degli Studi Di Trieste | Multifunctional polyethylene glycol derivatives: preparation and use |
WO2005123139A1 (en) | 2004-06-21 | 2005-12-29 | Universita' Degli Studi Di Trieste | Bifunctional derivatives of polyethylene glycol, their preparation and use |
EP1710249A1 (en) | 2004-01-27 | 2006-10-11 | Nippon Shinyaku Co., Ltd. | Ribonucleic acid compound and method of liquid-phase synthesis of oligonucleic acid compound |
US7276599B2 (en) | 2003-06-02 | 2007-10-02 | Isis Pharmaceuticals, Inc. | Oligonucleotide synthesis with alternative solvents |
US8143369B2 (en) | 2009-06-02 | 2012-03-27 | International Business Machines Corporation | Polymers bearing pendant pentafluorophenyl ester groups, and methods of synthesis and functionalization thereof |
US20130231260A1 (en) | 2012-02-17 | 2013-09-05 | NVS Technologies, Inc. | Polymer scaffolds for assay applications |
US8664357B2 (en) | 2008-08-08 | 2014-03-04 | Imperial Innovations Limited | Solvent resistant diafiltration of peptides, PNA or oligonucleotides |
US20140287945A1 (en) | 2013-03-14 | 2014-09-25 | NVS Technologies, Inc. | Surface oxidation for sequestering biomolecules and related methods |
WO2016160475A1 (en) * | 2015-03-30 | 2016-10-06 | Genapsys, Inc. | Beads for nucleic acid sequencing |
US20180023122A1 (en) | 2016-07-11 | 2018-01-25 | Glaxosmithkline Intellectual Property Development Limited | Novel processes for the production of oligonucleotides |
US20180100190A1 (en) | 2016-07-20 | 2018-04-12 | Genapsys, Inc. | Systems and methods for nucleic acid sequencing |
-
2021
- 2021-12-27 US US17/562,714 patent/US11725073B2/en active Active
Patent Citations (18)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
FR2623510A1 (en) | 1987-11-20 | 1989-05-26 | Ecole Nale Superieure Chimie | Polymers based on pentafluorophenyl halo- and methacrylates and their application to the production of optical fibres, of positive photoresists, of organic glasses and of resins for optical discs |
EP0850949A2 (en) | 1993-03-26 | 1998-07-01 | Institut Pasteur | Novel derivatives for use in nucleic acid sequencing |
US5798210A (en) | 1993-03-26 | 1998-08-25 | Institut Pasteur | Derivatives utilizable in nucleic acid sequencing |
WO2002079215A1 (en) | 2001-03-30 | 2002-10-10 | Isis Pharmaceuticals, Inc. | Building blocks for the solution phase synthesis of oligonucleotides |
US6677120B2 (en) | 2001-03-30 | 2004-01-13 | Isis Pharmaceuticals, Inc. | Building blocks for the solution phase synthesis of oligonucleotides |
WO2003093346A1 (en) | 2002-05-06 | 2003-11-13 | Universita' Degli Studi Di Trieste | Multifunctional polyethylene glycol derivatives: preparation and use |
US7276599B2 (en) | 2003-06-02 | 2007-10-02 | Isis Pharmaceuticals, Inc. | Oligonucleotide synthesis with alternative solvents |
EP1710249A1 (en) | 2004-01-27 | 2006-10-11 | Nippon Shinyaku Co., Ltd. | Ribonucleic acid compound and method of liquid-phase synthesis of oligonucleic acid compound |
WO2005123139A1 (en) | 2004-06-21 | 2005-12-29 | Universita' Degli Studi Di Trieste | Bifunctional derivatives of polyethylene glycol, their preparation and use |
US8664357B2 (en) | 2008-08-08 | 2014-03-04 | Imperial Innovations Limited | Solvent resistant diafiltration of peptides, PNA or oligonucleotides |
US8143369B2 (en) | 2009-06-02 | 2012-03-27 | International Business Machines Corporation | Polymers bearing pendant pentafluorophenyl ester groups, and methods of synthesis and functionalization thereof |
US8450504B2 (en) | 2009-06-02 | 2013-05-28 | International Business Machines Corporation | Polymers bearing pendant pentafluorophenyl ester groups, and methods of synthesis and functionalization thereof |
US20130231260A1 (en) | 2012-02-17 | 2013-09-05 | NVS Technologies, Inc. | Polymer scaffolds for assay applications |
US20140287945A1 (en) | 2013-03-14 | 2014-09-25 | NVS Technologies, Inc. | Surface oxidation for sequestering biomolecules and related methods |
WO2016160475A1 (en) * | 2015-03-30 | 2016-10-06 | Genapsys, Inc. | Beads for nucleic acid sequencing |
US20180023122A1 (en) | 2016-07-11 | 2018-01-25 | Glaxosmithkline Intellectual Property Development Limited | Novel processes for the production of oligonucleotides |
US20180100190A1 (en) | 2016-07-20 | 2018-04-12 | Genapsys, Inc. | Systems and methods for nucleic acid sequencing |
US10544456B2 (en) | 2016-07-20 | 2020-01-28 | Genapsys, Inc. | Systems and methods for nucleic acid sequencing |
Non-Patent Citations (23)
Title |
---|
Atdbio, 2021, Solid State Oligonucleotide Synthesis, https://www.atdbio.com/content/17/Solid-phase-oligonucleotide-synthesis, 26 pp. |
Beaucage et al., 1992, Advances in the synthesis of oligonucieotides by the phosphoramidite approach, Tetrahedron, 48(12):2223-2311. |
Bonora et al., Nucleic Acids Research, 1993, 21(5), p. 1213-1217. (Year: 1993). * |
Carey, 1992, Organic Chemistry, 2d ed., McGraw-Hill, Inc., New York, pp. 328-331. |
Creusen et al., 2020, Scalable one-pot—liquid-phase oligonucleotide synthesis for model network hydrogels, ChemRxiv., preprint. https://doi.org/10.26434/chemrxiv.12327569.v1. |
Gorelov et al., 1979, Thermal decomposition of poly(pheny/- and poly(pentafluorophenyl acrylates)), Vysokomoiekularnye Soedineniya, Seriya B: Bratkie Soobshcheniya, 21(6):410-413 (abstract). |
Gravert et al., Chem. Rev., 1997, 97, p. 489-509. (Year: 1997). * |
Greene et al., 1999, Protective Groups in Organic Synthesis, 3rd Ed., John Wiley & Sons, New York (TOC). |
Katayama et al., 2018, Liquid-phase synthesis of oligonucleotides, in Synthesis of Therapeutic Oligonucleotides, Obika et al., eds., Springer Nature Singapore Pte Ltd., pp. 83-95. |
Kim et al., 2013, Liquid-phase RNA synthesis by using alkyl-chain-soluble support, Chem. Eur. J., 19:8615-8620. |
Livingston, Jan. 2, 2020, Liquid phase oligonucleotide synthesis, Oxford Global, Biologies Series, 2 pp. |
McMurry, 2000, Organic Chemistry, 5th ed., Brooks/Cole, Pacific Grove, CA, pp. 398 and 408. |
McOmie, ed., 1973, Protective Groups in Organic Chemistry, Plenum Press (TOC). |
Merrifield, Jul. 20, 1963, Solid phase peptide synthesis. I. The synthesis of a tetrapeptide, J. Am. Chem. Soc., 85(14):2149-2154. |
Merrifield, Oct. 8, 1965, Automated synthesis of peptides: solid-phase peptide synthesis, a simple and rapid synthetic method, has now been automates, Science, 150(3693):178-185. |
Molina et al., 2019, Liquid-phase oligonucleotide synthesis: past, present, and future predictions, Current Protocols in Nucleic Acid Chemistry, 77:e82, 17 pp. |
Scheit, 1980. Nucleotide analogs: Synthesis and biological function. New York: John Wiley & Sons (TOC). |
Streitwieser et al., 1981, Introduction to Organic Chemistry, 2d ed., Macmillan Publishing Co., Inc., New York, pp. 169-171. |
Takahashi et al., 2012, Development of an efficient liquid-phase peptide synthesis protocol using a novel fluorene-derived anchor support compound with Fmoc chemistry; AJIPHASE®, Tetrahedron Lett., 53:1936-1939. |
Takahashi et al., 2012, Novel diphenylmethyl-derived amide protecting group for efficient liquid-phase peptide synthesis: AJIPHASE, Organic Lett., 14:4514-4517. |
Takahashi et al., 2017, AJIPHASE®: a highly efficient synthetic method for one-pot peptide elongation in the solution phase by an Fmoc strategy, Angew. Chem. Int. Ed., 56:7803-7807. |
Uhlman et al., Jun. 1990, Antisense oligonucleotides: a new therapeutic principle, Chemical Reviews, 90(4):543-584. |
Wang et al., Progress in Polymer Science, 2016, 53, p. 169-206. (Year: 2016). * |
Also Published As
Publication number | Publication date |
---|---|
US20220204670A1 (en) | 2022-06-30 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
US20240034748A1 (en) | Synthesis of Protected 3'-amino Nucleoside Monomers | |
US11059849B2 (en) | Modified nucleotides for synthesis of nucleic acids, a kit containing such nucleotides and their use for the production of synthetic nucleic acid sequences or genes | |
KR100782896B1 (en) | L-Ribo-LNA analogues | |
EP0191133B1 (en) | Stable salts of sam with polyanions | |
WO2001007455A1 (en) | Novel bicyclonucleoside analogues | |
US11214590B2 (en) | Photoresponsive nucleotide analog capable of photocrosslinking in visible light region | |
US11725073B2 (en) | Compositions and methods for liquid phase oligonucleotide synthesis | |
ES2553866T3 (en) | Polymers comprising a majority of amphiphilic monomers intended for the capture and manipulation of membrane proteins | |
US11851454B2 (en) | Compositions and methods for liquid phase oligonucleotide synthesis | |
EP1371733B1 (en) | Process for producing hyaluronic acid or its derivative | |
US20230212212A1 (en) | Processes for preparing oligonucleotides | |
EP0218190B1 (en) | Macromolecular cdp-choline derivatives, process for their preparation and pharmaceutical compositions containing them | |
CN110204583B (en) | Modified nucleoside, nucleotide and modified nucleic acid polymer as well as preparation method and application thereof | |
US20240002420A1 (en) | Compositions and methods for liquid phase oligonucleotide synthesis | |
KR102325668B1 (en) | Dithiolane functionalized nucleoside amidites and supports for stronger immobilization of bio-molecules on solid surfaces | |
US20230130509A1 (en) | Polymeric solid support for oligonucleotide synthesis | |
JP2002509155A (en) | Nucleobase oligomer | |
JP2014047170A (en) | Compound, polymer, surface treatment agent, and biocompatible material |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
FEPP | Fee payment procedure |
Free format text: ENTITY STATUS SET TO UNDISCOUNTED (ORIGINAL EVENT CODE: BIG.); ENTITY STATUS OF PATENT OWNER: SMALL ENTITY |
|
FEPP | Fee payment procedure |
Free format text: ENTITY STATUS SET TO SMALL (ORIGINAL EVENT CODE: SMAL); ENTITY STATUS OF PATENT OWNER: SMALL ENTITY |
|
STPP | Information on status: patent application and granting procedure in general |
Free format text: DOCKETED NEW CASE - READY FOR EXAMINATION |
|
STPP | Information on status: patent application and granting procedure in general |
Free format text: NON FINAL ACTION MAILED |
|
STPP | Information on status: patent application and granting procedure in general |
Free format text: RESPONSE TO NON-FINAL OFFICE ACTION ENTERED AND FORWARDED TO EXAMINER |
|
STPP | Information on status: patent application and granting procedure in general |
Free format text: NON FINAL ACTION MAILED |
|
AS | Assignment |
Owner name: HONGENE BIOTECH CORPORATION, CALIFORNIA Free format text: ASSIGNMENT OF ASSIGNORS INTEREST;ASSIGNORS:LAU, ALDRICH N.K.;GUAN, XIAOYANG;YU, DAVID;SIGNING DATES FROM 20010122 TO 20210122;REEL/FRAME:063209/0233 |
|
AS | Assignment |
Owner name: HONGENE BIOTECH CORPORATION, CALIFORNIA Free format text: ASSIGNMENT OF ASSIGNORS INTEREST;ASSIGNORS:LAU, ALDRICH N.K.;GUAN, XIAOYANG;YU, DAVID;REEL/FRAME:063307/0248 Effective date: 20210122 |
|
STPP | Information on status: patent application and granting procedure in general |
Free format text: PUBLICATIONS -- ISSUE FEE PAYMENT RECEIVED |
|
STCF | Information on status: patent grant |
Free format text: PATENTED CASE |
|
CC | Certificate of correction |